text:
Patent US8008353 - Alzheimer's disease; small molecule amine salts - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsMethods for modulating macrophage proliferation in an individual afflicted with or at risk for a macrophage-associated disease are provided. The methods employ a polyamine analog, or salt or protected derivative thereof. Macrophage proliferation has been implicated in a number of serious disorders, including...http://www.google.com/patents/US8008353?utm_source=gb-gplus-sharePatent US8008353 - Alzheimer's disease; small molecule amine saltsAdvanced Patent SearchPublication numberUS8008353 B2Publication typeGrantApplication numberUS 11/022,153Publication dateAug 30, 2011Filing dateDec 22, 2004Priority dateOct 27, 1997Also published asCA2308274A1, CA2308274C, DE69840048D1, EP1027040A2, EP1027040B1, US7087648, US20050159493, US20120022166, WO1999021542A2, WO1999021542A3Publication number022153, 11022153, US 8008353 B2, US 8008353B2, US-B2-8008353, US8008353 B2, US8008353B2InventorsMichael S. McGrathOriginal AssigneeThe Regents Of The University Of CaliforniaExport CitationBiBTeX, EndNote, RefManPatent Citations (33), Non-Patent Citations (34), Referenced by (2), Classifications (24), Legal Events (2) External Links: USPTO, USPTO Assignment, EspacenetAlzheimer's disease; small molecule amine saltsUS 8008353 B2Abstract Methods for modulating macrophage proliferation in an individual afflicted with or at risk for a macrophage-associated disease are provided. The methods employ a polyamine analog, or salt or protected derivative thereof. Macrophage proliferation has been implicated in a number of serious disorders, including AIDS (HIV)-associated dementia, AIDS-associated non-Hodgkin's lymphoma, and Alzheimer's disease. The invention also provides methods for aiding diagnosis and monitoring therapy of a macrophage-associated non-HIV associated dementia, especially Alzheimer's disease. The invention also provides methods of delaying development of macrophage-associated non-HIV associated dementias, including Alzheimer's disease, which entail administration of an agent which modulates macrophage proliferation.
1. A method of treating a subject afflicted with, or at risk for, HIV-associated dementia or delaying development or onset of HIV-associated dementia in said subject comprising administering to said subject effective amount of a composition comprising mitoguazone or a salt of mitoguazone, wherein the patient does not have AIDS-related non-Hodgkin's lymphoma.
2. The method of claim 1, wherein the composition comprises mitoguazone dihydrochloride.
3. A method of modulating macrophage proliferation in an individual afflicted with, or at risk for, HIV-associated dementia comprising administering to the individual an effective amount of a composition comprising mitoguazone or a salt of mitoguazone, wherein the patient does not have AIDS-related non-Hodgkin's lymphoma.
4. The method of claim 3, wherein the composition comprises mitoguazone dihydrochloride.
5. A method of modulating macrophage proliferation in an individual afflicted with, or at risk for, a macrophage-associated disease comprising administering to the individual an effective amount of a composition comprising mitoguazone or a salt of mitoguazone, wherein the disease is HIV-associated dementia or non-HIV-associated dementia, wherein the patient does not have AIDS-related non-Hodgkin's lymphoma.
6. The method of claim 5, wherein the composition comprises mitoguazone dihydrochloride.
7. A method of treating a subject afflicted with, or at risk for, a macrophage-associated disease or delaying onset or development of a macrophage-associated disease in said subject comprising administering to said subject an effective amount of a composition comprising mitoguazone or a salt of mitoguazone to reduce macrophage proliferation in said subject, wherein the disease is HIV-associated dementia or non-HIV-associated dementia, wherein the patient does not have AIDS-related non-Hodgkin's lymphoma.
8. The method of claim 7, wherein the composition comprises mitoguazone dihydrochloride.
CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 09/179,383, filed Oct. 26, 1998, which application claims the benefit of U.S. Provisional Application Ser. Nos. 60/063,317 and 60/063,318, both filed Oct. 27, 1997, each of which applications are incorporated by reference.
TECHNICAL FIELD This invention relates to macrophage proliferation. More specifically, it relates to the use of polyamine analogs or salts or protected derivatives thereof to modulate macrophage proliferation, particularly in individuals afflicted with or at risk for a macrophage-associated disease. The invention also relates to methods of aiding diagnosis monitoring therapy, and delaying development of macrophage proliferation disorders (in the context of non-HIV associated dementias, such as Alzheimer's disease) that entail detection and/or modulation of macrophage proliferation.
BACKGROUND ART Cell proliferative diseases represent a major health problem and threat worldwide. Such diseases are characterized by the single or multiple abnormal proliferation of cells, groups of cells or tissue(s). See U.S. Pat. No. 5,665,588. Cell proliferative diseases include, but are not limited to, AIDS dementia, various cancers, atherosclerosis, vitreoretinopathy, psoriasis, neurodegenerative disorders and nephropathy. See U.S. Pat. Nos. 5,639,600, 4,847,257 and 5,672,746. Dementias, or progressive mental failures, particularly present a serious worldwide health problem, both in terms of the extent of debilitation and health care costs. Dementias include non-HIV-associated dementias (such as Alzheimer's disease or AD) and HIV-associated dementia.
Macrophages are terminally differentiated cells generally incapable of further cell division. Surprisingly, macrophage proliferation has been implicated in certain serious proliferative diseases such as lymphoma, cardiovascular disease, and nephrosclerosis. U.S. Pat. No. 5,639,600. Gabrielian et al. reported the role of macrophage infiltration in traumatic proliferative vitreoretinopathy. (1994) Curr. Eye. Res. 13:1-9. McGrath et al. disclosed the involvement of clonally expanded macrophages in the induction of cancerous tumor growth and AIDS dementia. U.S. Pat. Nos. 5,639,600 and 5,580,715; see also Pulliam et al. (1997) Lancet 349:692-695; McGrath et al. (1995) J. Acquired Imm. Def. Syn. Hum. Retro. 8: 379-385; Shiramizu et al. (1994) Cancer Res. 54:2069-2072.
Several different classes of chemical compounds have been reported that can inhibit abnormal cellular proliferation. These classes include, for example, anionic oligomers, amino 1,2,3-triazoles, valproic acid derivatives and polyamines. Cardin et al. reported that certain anionic oligomers possess antiproliferative activity. U.S. Pat. No. 5,460,807. Water soluble polyureas and polyamides with a molecular weight of less than 10,000 inhibit smooth muscle cell proliferation in culture and in vivo. Cardin et al. state that the anionic oligomers can be used in the treatment of atherosclerosis. Hupe et al. disclosed that certain triazoles are antiproliferatives. U.S. Pat. No. 4,847,257. Amino 1, 2, 3 triazoles inhibit labeled thymidine incorporation into intact pig skin and also inhibit keratinocyte proliferation. Hupe et al. state that the triazoles can be used in the treatment of psoriasis, a chronic skin disease which is characterized by hyperproliferation of the epidermis Nau et al. discovered that certain acids inhibit cell mitosis. U.S. Pat. No. 5,672,746. Derivatives of valproic acid decrease neuro-2a cell proliferation in vitro. Nau et al. state that the valproic acid derivatives can be used for the prevention and treatment of neurodegenerative disorders such as Alzheimer's disease. The treatment would be aimed at preventing the adverse effects of the disease by directly inhibiting pathologic neural cell growth.
The level of polyamines is intimately related to cell proliferation. Cellular levels of polyamines are carefully regulated by opposing synthetic and catabolic pathways. Compounds that are able to lower polyamine levels are proposed for use in the treatment of rapidly proliferating host cells such as cancer and psoriasis. A key polyamine catabolizing enzyme spermidine-spermine N1-acetyltransferase (SSAT) is among the few genes known to be inducible by the natural polyamines. Certain polyamine analogs exaggerate this response. 1,11-diethylnorspermine (DENSPM) increases SSAT mRNA levels in human melanoma cells up to 20-fold, with an increase in immunodetectable SSAT protein by 300-fold. By comparison, natural polyamine spermine is far less effective, increasing SSAT mRNA by �3-fold and immunodetectable protein by �7-fold. Fogel-Petrovic et al. (1996) Biochemistry 35:14435. Polyamine analogs also induce Z-DNA structure in vitro. This property correlates inversely with the effects on cis-diaminedichloroplatinum (II) (CDDP) cytotoxicity in human brain tumor cells. Basu et al. (1996) Anticancer Res. 16:39.
U.S. Pat. No. 5,498,522 outlines the use of SSAT as a prognostic indicator or tumor response marker. Either SSAT enzyme activity, SSAT enzyme protein, or mRNA transcripts can be measured directly, or other determinants related to SSAT induction can be measured, such as SSAT co-factor acetylCoA, and the SSAT products N1-acetylspermine and N1-acetylspermidine. Measurement of these determinants is proposed as a prognostic indicia and tumor response marker to evaluate the clinical effectiveness of anticancer agents comprising polyamine analogs. Determination is performed by collecting tumor cells by biopsy before or after treatment, and measuring the selected determinant. Hibasami et al. [(1989) Cancer Res. 49:2065] prepared an inhibitor of the natural polyamine synthetic pathway. The compound, methylglyoxal-bis(cyclopentylamidinohydrazone) (MGBCP) inhibited S-adenosylmethionine decarboxylase, spermine synthase, and spermine synthetase, competing with S-adenosylmethionine, spermidine, and putrescine, respectively. MGBCP depleted spermidine and spermine in leukemic ascites cells, and prolonged survival time of mice bearing P388 leukemia.
U.S. Pat. No. 5,541,230 (Basu et al.) indicates that spermine derivatives decrease growth in a number of human tumor cell lines, and propose their use in cancer chemotherapy. Bergeron et al. (Cancer Chemother. Pharmacol.) showed that the polyamine analogs 1,14-bis(ethylamino)-5,10-diazatetradecaone (BE-4-4-4), and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) directly affects growth, survival, and cell cycle progression in human brain tumor cell lines. The synthesis of BE-4-4-4-4 is disclosed in U.S. Pat. No. 5,541,230. U.S. Pat. No. 5,516,807 (Hupe et al.) claims a method for treating vascular proliferative disorders following balloon angioplasty. Bis-ethyl norspermine was found to inhibit the growth of rat aortic smooth muscle cells in culture for 8 to 15 days. It is proposed that restenosis following balloon angioplasty, graft, shunt, or athereotomy can be treated or prevented by administration of an effective amount of a polyamine, although no such experiments were performed. For other publications relating to the synthesis and use of certain polyamines, the reader is referred to EP 277,635, EP 162,413, EP 399,519, JP 85/6348, and U.S. Pat. No. 5,679,682; and to Bellevue et al. (1996) Bioorg. Med. Chem. Lett. 6:2765, and Porter et al. (1992) Falk Symposium 62:201; Marton and Pegg (1995) Ann Rev. Pharmacol. Toxicol. 35:55-91.
DISCLOSURE OF THE INVENTION The present invention provides methods for modulating macrophage proliferation using a composition comprising a polyamine analog or salt or protected derivative thereof (or using a polyamine analog or salt or protected derivative thereof), preferably in an individual afflicted with or at risk for a disease with which macrophage proliferation is associated, wherein the composition (or compound) is administered in an amount sufficient to modulate macrophage proliferation. In one embodiment, the disease is a non-HIV-associated dementia, particularly Alzheimer's disease. In another embodiment, the individual is HIV-infected and the disease is HIV-associated, such as HIV-associated dementia. Preferably, all the nitrogens of the polyamine analog are secondary, tertiary, or quaternary amino groups. The composition can further comprise a pharmaceutically acceptable excipient. In some embodiments, the composition comprises mitoguazone dihydrochloride.
BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and 1B are graphs denoting percent inhibition or killing in vitro of macrophage proliferation by varying concentrations of polyamine analogs DEHOP (SunPharm) and BE-4444, compared to antineoplastic agent cyclosphosphamide. Blood was obtained from patients with AIDS dementia.
MODES FOR CARRYING OUT THE INVENTION We have discovered that polyamine analogs are particularly effective in modulating macrophage proliferation. We have also discovered elevated, abnormal levels of macrophage proliferation in peripheral blood of patients with Alzheimer's disease (AD). We have also observed that macrophage proliferation is associated with a number of other serious disorders, including AIDS dementia, AIDS-associated non-Hodgkin's lymphoma, cardiovascular disease and nephrosclerosis. U.S. Pat. No. 5,639,600; see also commonly owned U.S. provisional application U.S. Ser. No. 60/063,318. Without wishing to be bound by a particular theory, we note that this macrophage proliferation phenomenon is associated with, and may contribute to the disease sequelae of, non-HIV associated dementias such as AD.
As discussed below, preferred agents for modulation of macrophages are polyamine analogs especially 1,11-bis(ethyl)norspermine; 1,8-bis(ethyl)spermidine (BES); 1,12-bis(ethyl)spermine (BESm; DESPM (N1,N12-diethylspermine); 1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3); 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4) (Diethylhomospermine, N1,N14-diethylhomospermine; DEHOP or DEHSPM); diethyl-norspermine (DENOP); 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4); N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-cis-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride (SL-11037); N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-trans-cyclobutylmethyl)-propane 1,3-diamine tetrahydrochloride (SL-11038); N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-trans-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride (SL-11044; and N,N′-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4-diamine tetrahydrochloride (SL-11047).
A �macrophage-associated� disease, disorder or indication is a disease, disorder or indication that is associated with an elevated, or abnormal, level of macrophage proliferation as compared to control sample(s). Such disorders include, but are not limited to, AIDS-associated dementia, Alzheimer's disease (AD), AIDS lymphoma, follicular lymphoma, mycoses fungoides, T cell and B cell lymphomas with significant macrophage compartments, atherosclerosis, kidney disease such as focal segmental glomerulosclerosis and membrane proliferative glomerulo nephropathy, psoriaform dermatitities, AIDS-associated diarrhea, prelymphomatis autoimmune disease such as AILD (angioimmunoblastic lymphadenophathy with dysproteinemia) and herpes virus associated diseases such as Castleman's disease and Kaposi's sarcoma. The terms �disorder� and �disease� are used interchangeably herein. �Macrophage-associated dementia� is a dementia that is associated with an elevated, or abnormal, level of macrophage proliferation as compared to control sample(s). Such dementias include, but are not limited to, AD. A macrophage-associated disorder, disease or dementia can be HIV-mediated or non-HIV-mediated, or HIV-associated or non-HIV associated. A �non-HIV-mediated� disease or dementia is a disease or dementia which is not caused by HIV, either directly or indirectly. A �non-HIV-associated� disease or dementia is a disease or dementia not normally associated with or secondary to HIV infection. An �HIV-mediated� disease, dementia or indication is directly or indirectly caused by (and/or linked to) HIV infection. An �HIV-associated� disease, dementia or indication is defined more broadly as generally associated with or secondary to an HIV infection; �HIV-mediated� diseases, for example, are included in those considered to be �HIV-associated.�
An individual �afflicted with� a macrophage associated disorder or non-HIV associated dementia means that the individual individual has been diagnosed as having, or is suspected as having, a macrophage associated disoreder or a non-HIV associated dementia.
By �polyamine analog� is meant an organic cation structurally similar but non-identical to naturally-occurring polyamines such as spermine and/or spermidine and their precursor, diamine putrescine. Polyamine analogs can be branched or un-branched, or incorporate cyclic moieties. Examples of polyamine analogs include, without limitation, N1, N14-diethylhomo-spermine (DEHSPM) and N1,N12-diethylspermine (DESPM). See, for example, WO 98/17624 and U.S. Pat. No. 5,541,230. U.S. Pat. Nos. 5,037,846 and 5,242,947 disclose polyamines comprising primary amino groups. Especially preferred are polyamine analogs wherein all nitrogen atoms of said polyamine analogs are independently secondary, tertiary, or quartenary amino groups.
An �alkyl� is a cyclic, branched, or straight chain chemical group containing carbon and hydrogen, such as methyl, butyl, t-butyl, pentyl, cyclopropyl, and octyl. Alkyl groups can be either unsubstituted or substituted with one or more substituents, e.g., halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyl. Alkyl groups can be saturated or unsaturated (e.g., containing �C═C� or �C≡C� subunits), at one or several positions. Unless otherwise specified, alkyl groups will comprise 1 to 8 carbon atoms, preferably 1 to 6, and more preferably 1 to 4 carbon atoms. �Cycloalkyl� refers to cyclic alkyl groups only, such as cyclopropyl, cyclobutyl, cyclopentyl, etc. �n-alkyl� refers to a linear (i.e., straight-chain) alkyl group only, while �branched alkyl� refers to branched alkyl groups to the exclusion of cyclic and linear alkyl groups. �Alkenyl� refers to a cyclic, branched, or straight chain chemical group containing carbon and hydrogen where at least one bond is monounsaturated, such as ethenyl, cyclopentenyl, or 1,3-butadienyl. Alkenyl groups can be substituted as indicated for alkyl groups. Alkenyl groups can be designated as cyclic, linear (n-alkenyl) or branched in an analogous fashion to the preceding designations for alkyl. An �aryl� is an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl), or multiple condensed rings (e.g., naphthyl), which can optionally be unsubstituted or substituted with amino, hydroxyl, alkyl, alkoxy, chloro, halo, mercapto and other substituents.
As used herein, �biological sample� encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term �biological sample� encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. Generally, the sample will be, or be derived from, peripheral blood. Preferably, the blood will have been enriched for a macrophage fraction, by using, for example, glass or plastic adherence.
As used herein, �aiding diagnosis� means that these methods assist in making a clinical determination regarding the classification, or nature, of the dementia, and may or may not be conclusive with respect to the definitive diagnosis. The method of aiding diagnosis of a macrophage-associated disease can comprise the step of detecting the level of proliferating macrophages in a biological sample from the individual, wherein the disease is dementia, such as a non-HIV-associated dementia, such as Alzheimer's disease. Dementias may or may not be associated with clonal macrophage proliferation, and making this classification may assist in developing and recommending treatment strategies as well as evaluating prognosis.
�Development� of a dementia herein means initial manifestations and/or ensuing progression of the disorder. Development of disease can be detectable and assessed using standard clinical techniques, such as neurofunction/cognitive tests and brain scanning technologies such as MRI. However, development also refers to disease progression that may be undetectable. For purposes of this invention, progression refers to the biological course of the disease state. �Development� includes occurrence, recurrence, and onset. As used herein �onset� or �occurrence� of a neurological disorder includes initial onset and/or recurrence. As used herein, �delaying� development of disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disorder and/or the medical profile of the individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop detectable disease. A method that �delays� development of disease is a method that reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects, although this knowledge can be based upon anecdotal evidence. �Delaying development� can mean that the extent and/or undesirable clinical manifestations are lessened and/or time course of the progression is slowed or lengthened, as compared to not administering the agent. Thus the term also includes, but is not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, and remission (whether partial or total) whether detectable or undetectable.
�Modulating� macrophage proliferation means that the rate of proliferation is altered when compared to not administering an agent that interferes with natural polyamine interaction with DNA (including, but not limited to, interfering with a polyamine biosynthetic pathway, interfering with the intracellular concentration of spermidine, competitors, inhibitors of DNA interaction by a natural polyamine, interfering with polyamine metabolism, etc.), such as a polyamine analog. Preferably, �modulating� macrophage proliferation means a change in the rate of macrophage proliferation of at least 25%, preferably at least 50%, more preferably at least 75%, and even more preferably at least 90%. Generally, for purposes of this invention, �modulating� macrophage proliferation means that the rate of proliferation is decreased when compared to the rate of proliferation in that individual when no agent is administered. However, during the course of therapy, for example, it may be desirable to increase the rate of proliferation from a previously measured level. The degree of modulation may be assessed by measurement of macrophage proliferation, which will be discussed below, and generally entails detecting a proliferation marker(s) in a macrophage population or uptake of certain substances such as BrdU or 3H-thymidine (which would provide a quantitative measure of proliferation). Further, it is possible that, if the macrophages are proliferating due to a genetic alteration (such as transposition, deletion, or insertion), this alteration could be detected using standard techniques in the art, such as RFLP (restriction fragment length polymorphism).
For purposes of this invention, an individual suitable for administration of a polyamine analog (or, in certain contexts, such as non-HIV associated dementia other than AD, an agent which modulates macrophage proliferation) is one who has been diagnosed as having a macrophage-associated disorder, such as AIDS dementia, AIDS non-Hodgkin's lymphoma, Alzheimer's disease, or who is adjudged to be at high risk for developing such a disorder. In some embodiments, an individual suitable for administration of an agent which modulates macrophage proliferation is one who has been diagnosed as having a macrophage-associated, non HIV associated dementia other than AD, or who is adjudged to be at risk for developing such a dementia. As is evident to one skilled in the art, these methods can apply to those individuals not displaying any symptoms. An �at risk� or �high risk� individual is an individual who has a discrete and significant risk of developing disease (a macrophage-associated disorder). An �at risk� or �high risk� individual may or may not have detectable disease, and may or may not have displayed detectable disease prior to receiving the method(s) described herein. �High risk� (or �at risk�) denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of disease. An individual having one or more of these risk factors has a higher probability of developing disease than an individual without these risk factor(s). These risk factors include, but are not limited to, genetic (i.e., hereditary) considerations (including family history and genetic markers), and presence or absence of appropriate chemical markers and exposure to environments, conditions, or factors which would increase the possibility of acquiring a particular disease. Retroviral infections, especially retroviral insertions into particular genetic loci (such as fur or PDGF), may also be considered a risk factor. A high risk individual has one, preferably two, more preferably three, risk factors. However, it is understood that having only one risk factor can often indicate high risk.
Because all risk factors for developing macrophage-associated disease are not known, and the interplay among these factors (in terms of overall risk) are not fully understood, it is clear to one skilled in the art that individuals suitable for administration of an agent for purposes of this invention can have clinical features in common, and that individuals not falling clearly in the categories described above can nonetheless be considered suitable candidates for administration of an agent. For example, an individual having a genetic marker for development of a neurodegenerative disorder (such as the apoE gene) could be considered at risk for developing Alzheimer's disease, even though no previous disease has been observed. In this context, administration of an agent to such an individual could result in delay of occurrence of disease, even to the extent that the individual does not develop AD within his or her lifetime (or develops it later than would have been expected). Another example is an individual who is being treated using other modes of therapy, and who is showing clinical responsiveness to the therapy (i.e., stabilization or remission). Such an individual may be adjudged as at �high risk� even though the initial course of therapy is not yet completed, due to projection of clinical progress by the clinician, and can be a suitable candidate for receiving an agent before completion of the initial therapy. The clinician, as one skilled in the art, has discretion to determine whether treatment using an agent may be indicated.
In those individuals considered at high or significant risk of developing dementia, detection of proliferating macrophages in a biological sample may also assist in alerting the individual and/or the clinician of possible precursor disease. Thus, the invention also includes methods of monitoring an individual at risk or high risk of developing dementia, comprising detection of proliferating macrophages in a biological sample from that individual. Preferably, the individual is �afflicted with� (e.g., diagnosed as having, suffering from and/or displaying one or more clinical symptoms of) a particular disease, disorder or indication, or at �risk� for (e.g., having a genetic predisposition for, or family history of, or being environmentally exposed to factors which increase the probability of acquiring) a particular disease, disorder, or indication.
Detection of proliferating macrophage(s) can be achieved using any of several techniques. In some embodiments of the invention, proliferation is measured in relation to circulating macrophages, and is performed on a leukocyte preparation from peripheral blood. In other embodiments of the invention, proliferation is measured in relation to tissue-fixed macrophages, typically performed on tissue sections. Proliferating macrophages may be detected, for example, by assaying cell proliferative markers, such as PCNA, Ki67 or uptake of bromodeoxyuridine (BrdU) or 3H-thymidine. These markers are distinct from those that identify only �activated� macrophages (as opposed to proliferating macrophages), such as CD69 and CD25. The cellular subset representing macrophages may in turn be identified by detection of certain cell specific markers, such as CD14, CD68, CD16, or nonspecific esterase. Detection of these cell-type and/or proliferative markers use methods standard in the art, such as staining techniques and FACS sorting and analysis. These methods are further described in Example 1. Further, it is possible that these proliferating macrophages could be distinguished based on other characteristics, such as cell density (as measured in PERCOLL� gradients, for example). These determinations may be established empirically using standard techniques in the art.
where R2, R3, R4 and R5 are independently selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C1-C6 alkyl-C3-C6 cycloalkyl-C1-C6 alkyl C3-C10 aryl, and C1-C6 alkyl-C3-C10-aryl-C1-C6 alkyl;
More preferably, the polyamine analogs will include compounds of the structures 2 and 3, where R3, R5, R7 and R9 are independently (CH2), groups, where x is an integer from 2 to 6, and where R4, R6 and R8 are hydrogen atoms, and where R1 and R10 are alkyl groups, and further where R2 and R11 are hydrogen atoms.
where R4 is C2-C6 n-alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, or C3-C6 aryl;
Preferably, all the nitrogens of the polyamine analog are independently secondary, tertiary, or quaternary amino groups.
1,11-bis(ethyl)norspermine (1,11-bis(ethylamino)-4,8-diazaundecane; BE-3-3-3) 1,8-bis(ethyl)spermidine (BES) 1,12-bis(ethyl)spermine (BESm; DESPM (N1,N12-diethylspermine; SunPharm); 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4) (Diethylhomospermine, N1,N14-diethylhomospermine; DEHOP or DEHSPM; SunPharm) diethyl-norspermine (DENOP; SunPharm) 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-cis-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride (SL-11037), provided by S'LIL, Madison, Wis. N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-trans-cyclobutylmethyl)-propane 1,3-diamine tetrahydrochloride (SL-11038), S'LIL N-ethyl-N′-(2-(3′-ethylamino-propylamino methyl)-trans-cyclopropylmethyl)-propane 1,3-diamine tetrahydrochloride (SL-11044), S'LIL. N,N′-bis(3-ethylaminopropyl)-cis-but-2-ene-1,4-diamine tetrahydrochloride (SL-11047), S'LIL
Besides the polyamine analogs listed above, stereoisomers, salts or protected derivatives thereof, may be used. The invention also comprises methods of using an effective amount of any of the polyamine analogs listed above, or stereoisomers, salts or protected derivatives thereof (or a composition comprising an effective amount of any of the polyamine analogs listed above, or stereoisomers, salts or protected derivatives thereof) in modulating macrophage proliferation (or in treating or delaying development of macrophage-associated diseases, including HIV-associated dementia and AD). The invention also comprises any polyamine analog listed above, or stereoisomers, salts or protected derivatives thereof, for use in preparing compositions (i.e., medicaments) useful for treating macrophage-associated diseases, including HIV-associated dementia, and Alzheimer's disease.
A polyamine analog (or stereoisomer, salt or protected derivative) may be administered alone, or in conjunction with other substances and/or therapies, depending on the context of administration (i.e., desired end result, condition of the individual, and indications). �In conjunction with� means that an agent is administered prior to, concurrently, or after other substance or therapy. Examples of substances that might be administered in conjunction with an agent include, but are not limited to, brain neurochemical modulators (in the context of macrophage-associated dementias), and classic anti-neoplastic agents and/or anti-lymphocytic agents such as steroids and cyclosporine derivatives. For example, a polyamine analog (or a stereoisomer, salt or protected derivative thereof) can be administered in conjunction with mitoguazone dihydrochloride.
The mechanistic effectiveness of various polyamine analogs and enzyme inhibitors can be determined at least in part by their ability to deplete intracellular polyamine pools. Kramer et al. [(1995) Biochem. Pharmacol. 50:1433[describe the use of 4-fluoro-L-ornithine to monitor metabolic flux through the polyamine biosynthetic pathway. It was determined that the metabolic flux indicated by the rate of appearance of fluorinated polyamines, reflected the proliferation status of the cells. U.S. Pat. No. 5,498,522 outlines the use of SSAT as a prognostic indicator or tumor response marker. Either SSAT enzyme activity, SSAT enzyme protein, or mRNA transcripts can be measured directly, or other determinants related to SSAT induction can be measured, such as SSAT co-factor acetylCoA, and the SSAT products N1-acetylspermine and N1-acetylspermidine. To further determine the effect of a polyamine analog's administration, an individual may be monitored for disease (or precursor disease) progression as well as biochemical and/or genetic markers of disease (or precursor disease). With respect to disease progression, multiple rating scales (i.e., indices of clinical function) have been established and are known in the art for various macrophage proliferative disorders such as AD and lymphomas. For macrophage-associated neurological disorders, cognitive functions can be tested and, in some cases, imaging modalities such as MRI may be used.
EXAMPLES Example 1 Testing Various Polyamine Analogs for Effect on Macrophage Proliferation in the Context of Macrophage-Associated Disorders Separation of Peripheral Blood Monocytes
Five�105 PBMC's isolated through a 1.087 g/cc Percoll/saline gradient were exposed to varied concentrations of agent after baseline CD14/PCNA staining was performed. The cells were cultured at 5�105 cells in RPM1-1640/10% fetal calf serum in a polypropylene tube (Falcon) at 37� C. for five days. After five days CD14/PCNA staining was performed on control and agent-treated cultures. FACSCAN analysis of PCNA-positive cells in control cultures was compared with agent-treated cultures and the percentage of control PCNA/CD14 cells was calculated. The results of such an experiment in which the effect of polyamine analog DEHOP (SunPharm; Florida) on proliferating macrophages (as detected by PCNA) from the blood of four patients with AIDS dementia are shown in FIG. 1A (with underlying data provided in Table 3). This polyamine analog significantly reduced the percentage of proliferating macrophages. A comparison of the inhibitory effect of DEHOP and cyclosphosphamide, another anti-proliferative agent, was performed in vitro using the blood of one of these patients (�CB�). The results are shown in Table 1. This experiment showed that DEHOP was superior in inhibition. This polyamine analog effect is relative and specific as indicated by a lack of in vitro toxicity in this assay system by cyclosphosphamide, a classic anti-neoplastic agent.
A comparison of polyamine analogs SL-11037, SL-11038 (S'LIL Pharmaceuticals) and DENOP (SunPharm) on macrophage proliferation from the blood of one patient with AIDS dementia is shown in Table 2. All 3 agents showed inhibitory effects. FIG. 1B similarly shows that polyamine analog BE-4444 demonstrates potent killing of PCNA+/CD14+ cells. The data shown in FIG. 5 similarly demonstrate that polyamine analogs SL-11047, SL-11048 and SL-11044 (S'LIL Pharmaceuticals) are potent inhibitors of PCNA+ macrophage detection from peripheral blood of HIV+ sarcoid patient. 60% of input CD14 cells were PCNA+; the assay was performed 96 hr after drug exposure and culture. The data shown in FIG. 6 demonstrate that polyamine analogs DEHOP and SL-11047 all decrease PCNA+/CD14 cell survival. The data presented are averages of several (N) experiments performed. Thus, the data shown herein demonstrate that BE-4444, SL-11037, SL-11038, SL-11044, SL-11047, SL-11048, DEHOP and DENOP all demonstrated potent inhibition and/or killing of blood PCNA+/CD14+ cells.
Effect of DEHOP and Cyclophosphamide (Cytoxan �, Bristol-
Myers Squibb) on CD14+, PCNA+ peripheral blood macrophage
Patient CB at time 0:
% of CD14+ that were CD69+: 0
% of CD14+ that were PCNA+: 28%
median SSC for all CD14+: 420
Example 2 Suppression of In Vitro Cell Killing by Supernatants from Peripheral Blood from an AIDS Dementia Patient We have previously observed a cell killing effect of mononuclear cell supernatants from peripheral blood of patients with AIDS dementia. Pulliam et al. (1997) Lancet 349: 692-695. We tested a similarly prepared supernatant which was treated with the polyamine analog DEHOP.
Human fetal brain tissue from between 16 and 18 weeks' gestation was obtained from elective abortions for preparation of brain cell aggregates. These aggregates contain all the cells of the central nervous system including neurons, astrocytes, oligodendrocytes with accompanying myelin, and microglial cells. After 10 days in culture, and before experimentation, aggregates were tested for viability by trypan-blue exclusion. Aggregates were divided into flasks at 50 mg wet-weight in 2 mL exchange medium (DMEM with 15% fetal calf serum). Aggregates were treated with mononuclear cell supernatants (20%) for 3 days. The flasks contents were centrifuged; the supernatant was used for lactate dehydrogenase assay and the aggregate pellet for DNA fragmentation (programmed cell death) assay.
Brain cell aggregates were treated with 20% supernatant from cultured CD14 cells from a demented AIDS patient. Treatment was for 72 h. After treatment, the aggregates were washed and fixed in Karnovsky's solution. The cells were post-fixed in 1% osmium tetroxide dehydrated for staining with uranyl acetate and lead citrate. Thin sections were examined in a JEOL 1OOSX electron microscope.
Example 3 Detection of Proliferating Macrophages in Patients with Alzheimer's Disease Participants and Methods
5 mL blood was obtained from patients and controls. 100 μL whole heparinised blood was stained with fluorescein-conjugated anti-CD14 (DAKO Corp.) and with phycoerythrin-conjugated anti-PCNA (Becton Dickinson). Red-blood cells were lysed by the addition of FACSLYSE solution (Becton Dickinson). The cell suspensions were centrifuged and the cell pellets resuspended in phosphate-buffered saline containing sodium azide and paraformaldehyde and stored at 4� C. To stain intracellularly for PCNA, cell pellets were resuspended in �Permeabilising Solution� (Becton Dickinson). Buffer was added to each tube, followed by centrifugation. Cell pellets were resuspended in buffer and stained with phycoerythrin-conjugated PCNA. The cells were washed, fixed, and stored as above.
Cells were analysed with a FACSCAN flow cytometer (Becton Dickinson). At least 2000 cells per sample were analysed. Negative staining was defined as the area of dot plots that contained over 99% of isotype-stained (DAKO) �control cells�. The results are shown in FIG. 4. The patients with AD displayed a significantly higher percentage of proliferating macrophages or non-disease controls as well as diseased controls, including AIDS dementia. Individual values (percentage PCNA/CD14 cells) were: (a) non-disease controls, 10; 16; 19; 20; 27; 46; (b) disease controls: ALS, 17; 25; Parkinson's, 27; 45; MS, 7; 20; HIV dementia, 6; 28; 37; 52; 54; 61; 96; (c) AD patients, 44; 47; 91; 97; 98; 100.
Example 4 Effect of Polyamine Analogs on PCNA Expression of Peripheral Macrophages from Patients with AD Separation of Peripheral Blood Monocytes
Five�105 PBMC's isolated through a 1.087 g/cc Percoll/saline gradient were exposed to varied concentrations of agent after baseline CD14/PCNA staining was performed as described in Example 3. The cells were cultured at 5�10 cells in RPM1-1640/10% fetal calf serum in a polypropylene tube (Falcon) at 37� C. for five days. After five days CD14/PCNA staining was performed on control and agent-treated cultures. FACSCAN analysis of PCNA-positive cells in control cultures was compared with agent-treated cultures and percentage of control PCNA/CD14 cells was calculated. Two polyamine analogs, DENOP (SunPharm; Florida) and SL-11037 were tested. As shown in FIG. 3, both agents significantly reduced the percentage of proliferating macrophages.
Example 5 Using Polyamine Analog to Treat an Individual with a Macrophage-Associated Dementia A 72-year-old male presents with increasing loss of memory and confusion. A blood sample is obtained, and the percentage of PCNA-expressing CD14 cells is significantly elevated over the control. The patient is given DESPM (a polyamine analog) at 125 mg/m2/day for six days, repeated every three weeks. Blood samples are obtained routinely to monitor the level of PCNA-expressing CD14 cells.
Example 6 Effects of Polyamine Analog Diethylhomospermine (DEHOP) on Chemotherapy Resistant AIDS Related Lymphoma Patients Four patients who had failed standard chemotherapy for treatment of their AIDS related lymphoma (therapy included at least CHOP chemotherapy up to six cycles) were evaluated for response to DEHOP administration in vivo. Patients had blood drawn pre administration of drug for evaluation of macrophage activation. This blood was also evaluated for response to DEHOP in vitro. The trial was set up to evaluate clinical response to drug given for 5 days subcutaneously (sq) at a dose of 25 mg/m2 (2 patients) and 50 mg/m2 (2 patients). Blood was obtained at 1 week after completion of the first and second cycles of therapy. The blood specimens were evaluated for macrophage proliferation (proliferating cell nuclear antigen, PCNA expression), and activation (percentage CD69 expression). Response to therapy was evaluated by direct observation of tumor (3 patients) and persistence or resolution of symptoms (1 patient with GI lymphoma).
To test whether patient macrophages would respond to administration of DEHOP in vitro, CD14 cells in a peripheral blood mononuclear cell preparation (PBMC) were exposed to different concentrations of DEHOP and the proportion of CD14 positive cells surviving at the end of 5 days of culture was assessed in drug treated cells as compared to controls. Specimens obtained pre chemotherapy were compared to specimens obtained 2 weeks post therapy. Table 4 summarizes CD14+ cell responses to DEHOP in vitro in the 4 AIDS lymphoma patients evaluated; FIG. 7 shows the CD14+ cell response to DEHOP in vivo. For patient FL, the first specimen was obtained one day after the first cycle of DEHOP; the second specimen was obtained one week later. The effective dose 50 (where 50% of cells were killed), and effective dose 80 (where 80% of cells were killed) were calculated for each set of patient specimens exposed to DEHOP in vitro. In post in vivo DEHOP administration blood specimens, patient FTJ was persistently resistant to drug whereas patient LT and FL, both of whom responded clinically to DEHOP, showed marked drug sensitivity in vitro.
CD14 Cell Polyamine Analog Sensitivity: In vitro dose response in NHL patient PBMC; total CD14+ cell survival, ED50 and ED80 Patient
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS4201788Aug 11, 1978May 6, 1980University Patents, Inc.Process for alleviating proliferative skin diseasesUS4847257Aug 20, 1987Jul 11, 1989Merck & Co., Inc.PsoriasisUS5037846Jul 31, 1990Aug 6, 1991Pfizer Inc.Found in spider venom; treatment of strokes, alzheimer's disease, epilepsyUS5091576Jun 23, 1988Feb 25, 1992University Of FloridaAntipsoriasis, insecticidesUS5242947Jan 3, 1992Sep 7, 1993New York UniversityUse of polyamines as ionic-channel regulating agentsUS5460807Jun 15, 1993Oct 24, 1995Merrell Dow Pharmaceuticals Inc.Polyurea or polyamide oligomers useful for inhibiting smooth muscle cellsUS5498522Nov 16, 1993Mar 12, 1996Health Research, Inc.Methods for the use of spermidine/spermine N1 -acetyltransferase as a prognostic indicator and/or a tumor response markerUS5516807Oct 25, 1994May 14, 1996Warner-Lambert CompanyAdministering a polyamine to a patient to treat or prevent restenosisUS5541230Nov 5, 1993Jul 30, 1996Us HealthAntitumor agentsUS5580715Aug 5, 1994Dec 3, 1996The Regents Of The University Of CaliforniaDiagnosis of cancer having clonal macrophage involvementUS5639600Jun 6, 1995Jun 17, 1997The Regents Of The University Of CaliforniaDiagnosis and treatment of cell proliferative disease having clonal macrophage involvementUS5665588Mar 2, 1995Sep 9, 1997Univ ArkansasDNA encoding natural killer lytic associated proteinUS5672746Aug 30, 1994Sep 30, 1997American Biogenetic Sciences, Inc.Antiproliferative and neurotrophic moleculesUS5677350Jun 7, 1995Oct 14, 1997Wisconsin Alumni Research FoundationInhibition of cancer cell growth, proliferation, and metastasis using N,N'-dα,ω-diaminoalkanesUS5679682Sep 18, 1996Oct 21, 1997University Of Florida Research Foundation, Inc.Anti-neoplastic, anti-viral or anti-retroviral spermine derivativesUS5880161Jul 29, 1996Mar 9, 1999The United States Of America As Represented By The Department Of Health And Human ServicesTherapeutic polyaminesUS5886051Nov 8, 1995Mar 23, 1999University Of Florida Research Foundation, Inc.Methods and compositions for the treatment of neurodegenerationUS5889061Oct 15, 1997Mar 30, 1999Wisconsin Alumni Research FoundationConformationally restricted polyaminesUS6392098Mar 29, 1999May 21, 2002Wisconsin Alumni Research FoundationAnticancer agentsEP0162413A2May 15, 1985Nov 27, 1985Merrell Dow Pharmaceuticals Inc.Polyamine for use as a medicine in the treatment of neoplasmsEP0277635A2Feb 2, 1988Aug 10, 1988Merrell Dow Pharmaceuticals Inc.Novel polyamine derivativesEP0399519A2May 23, 1990Nov 28, 1990Merrell Pharmaceuticals Inc.Potentiating cell-mediated immunity utilizing polyamine derivativesEP0415801A1Jun 26, 1990Mar 6, 1991N.V. Innogenetics S.A.Monoclonal antibodies against activated microglial cellsEP0436332A2Dec 14, 1990Jul 10, 1991Pfizer Inc.Polyamines useful as antagonists of excitatory amino acid neurotransmittersJPH08217670A Title not availableJPS606348A Title not availableWO1992013548A1Jan 31, 1992Aug 20, 1992Immunex CorpMethod of inhibiting replication of hiv in macrophagesWO1993004036A1Aug 10, 1992Mar 4, 1993Nps Pharma IncSynthetic aryl polyamines as excitatory amino acid neurotransmitter antagonistsWO1993014782A1Jan 28, 1993Aug 5, 1993Jolla Cancer Res FoundMethods of controlling the proliferation of macrophagesWO1996004019A1Aug 1, 1995Feb 15, 1996Univ CaliforniaDiagnosis and treatment of cell proliferative disease having clonal macrophage involvementWO1997033175A1Mar 3, 1997Sep 12, 1997Hartwig ManfredProcess for diagnosing alzheimer's disease and agents for use in therapyWO1998017624A1Oct 15, 1997Apr 30, 1998Wisconsin Alumni Res FoundConformationally restricted polyamines and their use as antineoplastic agentsWO1999003823A2Jul 15, 1998Jan 28, 1999Donald E BergstromNovel polyamine analogues as therapeutic and diagnostic agentsNon-Patent CitationsReference1Basu et al. "The Ability of Polyamine Analogues to Induce Z-DNA Structure in Synthetic Ploynucleotides in Vitro in Inversely Correlates with their Effects on Cytotoxcity of Cisdiaminedichloroplatinum (II) (CDDP) in Human Brain Tumor Cell Lines" (1996) Anticancer Res. 16:39-48.2Bellevue , III et al. "Structural Comparison of Alkylpolyamine Analogues with Potent in Vitro Antitumor or Antiparasitic Activity" (1996) Bioorg. Med. Chem. Lett. 6:2765-2770.3Bergeron et al. "Two Polyamine Analogs (BE-4-4-4 and BE-4-4-4-4) Directly Affect Growth, Survival, and Cell Cycle Progression in Two Human Brain Tumor Cell Lines" (1995) Cancer Chemother. Pharmacol. 36:411-417.4Bersnstein et al. "The Cellular Localization of the L-Orinthine Decarboxylase Polyamine System in Normal and Diseased Central Nervous Systems," Progress in Neurobiology 57(5):485-505, 1999.5Bitonti et al. "Bis(benzyl)poluamine Analogs Inhibit the Growth of Chloroquine-Resistant Human Malaria Parasites (Plasmodium falciparum) In Vitro and In Combination With alpha-Difluoromethylomithine Cure Murine Malaria" (1989) Proc. Natl. Acad. Sci. USA 86:651-655.6Bitonti et al. "Bis(benzyl)poluamine Analogs Inhibit the Growth of Chloroquine-Resistant Human Malaria Parasites (Plasmodium falciparum) In Vitro and In Combination With α-Difluoromethylomithine Cure Murine Malaria" (1989) Proc. Natl. Acad. Sci. USA 86:651-655.7Chiang et al. "Antihuman Immunodeficiency Virus (HIV-1) Activities of Inhibitors of Polyamine Pathways," J. Biomed. Sci. 3(2): 78-81, 1996.8Fields et al., "Fields Virology," vol. 2, 1996, Lippincott Williams & Wilkins, pp. 2418-2419.9Fogel-Petrovic et al. "Effects of Polyamines, Polyamine Analogs, and Inhibitors of Protein Synthesis on Spermidine-Spermine N'-Acetyltransferase Gene Expression," (1996) Biochemistry 35:14436-14444.10Fogel-Petrovic et al. "Effects of Polyamines, Polyamine Analogs, and Inhibitors of Protein Synthesis on Spermidine-Spermine N′-Acetyltransferase Gene Expression," (1996) Biochemistry 35:14436-14444.11Gabrielian et al., "Effect of Leukopenia on Experimental Post-Traumatic Retinal Detachment" (1993) Curr. Eye Res. 13:1-9.12Greene et al., Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, Inc. New York. (Table of Contents) (1991).13Hibasami et al., "Antitumor Effect of a New Multienzyme Inhibitor of Polyamine Synthetic Pathway, Methyglyocal-Bis(Cyclopentylamidinohydrazone), Against Human and Mouse Leukemia Cells" (1989) Cancer Res. 49:2065-2068.14Kackzmarek et al., "Inhibitors of Polyamine Biosynthesis Block Tumor Necrosis Factor-Induced Activation of Macrophages," Cancer Res. 52(7):1891-1894, 1992.15Kramer et al., "Use of 4-Fluoro-l-ornithine to Monitor Metabolic Flex Through The Polyamine Biosynthetic Pathway" (1995) Biochem. Pharmacol. 50:1433-1443.16Lim et al., "MGBG Therapy of Relasped Extralymphatic HIV-Associated Non-Hodgkins Lymphoma (HIV NHL)," (Mar. 14, 1995) Proc. Annu. Meet. Am. Soc. Clin. Oncol. 14:A1274.17Marton et al., "Polyamines As Targets for Therapeutic Intervention" (1995) Ann Rev. Pharmacol. Toxicol. 35:55-91.18Mascolini. "Oncologists Scout New Direction for KS and Lymphoma Therapies," (1995) J. Int. Assoc. Physicians Aids Care 1(5):10-14.19McGrath et al., "Indentification of A Clonal Form Of HIV In Early Kaposi's Sarcoma: Evidence For A Novel Model Of Oncogenesis, 'Sequential Neoplasia'"(1995) J. Acquired Inn. Def. Syn. Hum. Retro. 8:379-385.20McGrath et al., "Indentification of A Clonal Form Of HIV In Early Kaposi's Sarcoma: Evidence For A Novel Model Of Oncogenesis, �Sequential Neoplasia�"(1995) J. Acquired Inn. Def. Syn. Hum. Retro. 8:379-385.21Messina et al., "Polyamine Involvement In Functional Activation of Human Macrophages," (1992) J. Leukoc. Biol. 52(6):585-587.22Miles et al., "Curative Therapy of AIDS Cancers," (Jul. 1, 1996) Int. Conf. AIDS. XI(1):28.23Mukhopadhyay et al., "Effects of Bis(Benzl)Polyamine Analogs On Leishmania Donavani Promatigotes," (1995)Exper. Parasitology 81:39-46.24Pennings et al., "Sensitization of TNF-Induced Apoptosis With Polyamine Synthesis Inhibitors in Difference Human and Murine Tumor Cell Lines,"Cytokine 10(6):423-431, 1998.25Porter et al., "Polyamine Inhibitors and Analogues As Potential Anticancer Agents," (1992) Falk Symposium 62-Polyamines In The Gastrointestinal Tract Chapter 31, pp. 301-322, 1992.26Prakash et al., "Antitumor Activity of Norspemidine, A Structural Homologue of the Natural Polyamine Spermindine," Anticancer Research 8:563-568 (1998).27Pulliam et al., "Unique Monocyte Subset in Patients with AIDS Dementia," (1997) Lancet 349:692-695.28Reminton's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990) (Table of Contents).29 *Rizzo et al. "Pharmacokinetic profile of mitoguazone (MGBG) in patient with AIDS related non-Hodgkin's lymphoma," Investigational New Drugs, 1996, vol. 14, pp. 227-234.30Rizzo et al., "Pharmacokinetic Profile of Mitoguazone (MGBG) In Patients With AIDS Related Non-Hodgkins Llymphoma," (1996), Inventigational New Drug, vol. 14, pp. 227-234.31Shiramizu et al., "Identification of a Common Clonal Human Immunodeficiency Virus Integration Site in Human Immunodeficiency Virus-Associated Lymphomas", (1994) Cancer Res. 54:2069-2072.32 *Von Hoff "MGBG: Teaching an old drug new tricks," Annals of Oncology, 1994, vol. 5, pp. 487-493.33Von Hoff, "MGBG: Teaching an Old Drug New Tricks," (1994), Annals of Oncology, vol. 5. pages 487-493.34Wojewodzka et al., "Structure-Activity Relationship of Polyamine Derivatives of 1, 3-Dichloracetone-Thiosemicarbazone: Induction of Metastases and Increase In Sialylation of Murine Lymphoma L5178Y-R Cells," Chem. Biol. Interactions, 74 (1990) 221-223.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS20110091418 *Dec 21, 2010Apr 21, 2011Pathlogica, LLCMethods for treating viral infections using polyamine analogsUS20120022166 *Jul 25, 2011Jan 26, 2012Mcgrath Michael SMethods for Modulating Macrophage Proliferation Using Polyamine Analogs* Cited by examinerClassifications U.S. Classification514/635, 514/634International ClassificationA61P43/00, G01N33/569, A61K31/131, G01N33/50, A61K31/132, A61K31/155, A61K31/13, G01N33/68, A61P25/28Cooperative ClassificationA61K31/155, G01N2800/2821, G01N33/6896, A61K31/13, A61K31/131, G01N33/56972, G01N33/5055European ClassificationG01N33/50D2F2F, G01N33/68V2, A61K31/131, G01N33/569H2, A61K31/155, A61K31/13Legal EventsDateCodeEventDescriptionMar 6, 2012CCCertificate of correctionJun 20, 2005ASAssignmentOwner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIFFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MCGRATH, MICHAEL S.;REEL/FRAME:016162/0495Effective date: 19981218RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google

value: Google Patents is a search engine from Google that indexes patents and patent applications.


text:
Ok, I haven't been blogging anything of substance lately, but I haven't been on the internet much lately. I have to confess, we haven't been doing well at taking pictures or videos of the girls lately. What we do have, I need to download to the computer. Maybe after this trip to Yokota I can sit down and get that taken care of. By the way, we are leaving for Yokota AB tonight. We'll stay for two nights, get shots for our upcoming trip to Vietnam, and Eric will take his last ACSC test.
We haven't done much the last month that warrants a good blog. Days are filled with me taking care of the girls and going to parks. Eric says I have been spending my days sculpting the minds of our two daughters who will hold high ranks in the world, maybe even president. I look at my girls and say there is no way that will ever happen! Anyway, the most exciting video we have is of Chloe eating ketchup straight. So, we'll save you from that (for now) until we have something worth putting on here. I'll try getting some good pictures of the girls up soon; I know my parents live to see new pictures of the girls.
Pray for a safe three hour drive to Yokota AB this evening as well as our return Wednesday. Hopefully no throwing up or constant screaming fits. Also, we are staying in a one bedroom room versus the normal three bedroom. Last time it didn't go well so pray for a better result this time (aka the girls actually sleep).
I have been feeling the same way about having nothing really interesting to blog about. Is it bad to say I hope your Yokota trip is just as uneventful? Hmmmmm. Well, buy some yummy American food for them and it will all get better from there. :)

type: global

value: Blogger is an American online content management system (CMS) which enables multi-user blogs with time-stamped entries. Pyra Labs developed it before being acquired by Google in 2003.


text:
Sonesta ES Suites Orlando | Groupon
Sonesta ES Suites Orlando - Orlando, FL
Top Reasons to Visit Sonesta ES Suites Orlando
Located in the International Drive Resort Area, the hotel is within walking distance of many restaurants, shops, and outdoor activities.
A free shuttle service makes stops at the area’s biggest attractions, including Walt Disney World® Theme Parks, Universal Studios Orlando, and SeaWorld.
Each suite is outfitted with a full kitchen, dining area, and living room. The spacious layouts of one- two, and trio two-bedroom units are ideal for families.
A complimentary breakfast is served each morning in the common room and includes a variety of pastries, steel-cut oatmeal, and a make-your-own-waffle station.
Locally crafted beers and wine from area vineyards are available at the lobby bar, and there’s a daily happy hour from 5:30 p.m. to 7 p.m.
Stay for four, six, or eight, with dates through August
Credit card required at check-in to place a hold for incidentals
By plane: 14 miles from Orlando International Airport (MCO)
By cab: about $40 from MCO, including tip
The hotel is set to undergo renovations beginning in late August. The hotel will still remain open during this time, but some areas will be closed while the guest rooms and common areas are rebuilt. During renovations, the hotel will have a temporary entrance and check-in area and a continental breakfast will be served in a meeting room with limited seating.
very spacious wonderful wonderful staff!!!!! Everything was spectacular if I could go again I would definitely stay at sonesta inn suites.. they r remodeling their rooms in October and we were able to take a sneak peak at the new models and I have to say they are absolutely a jaw dropper.. I recommend this hotel to everyone!!! Also great breakfast....
Enedina M. · September 19, 2013
Great hotel! Will be back soon.
Adela W. · September 15, 2013
I dont think there is just one best thing. This hotel was great. It had a friendly staff and was clean from rooms to lobby to outside. Also breakfast buffet was different every morning, great for those who are staying multiple days!

value: Groupon is an American global e-commerce marketplace connecting subscribers with local merchants by offering activities, travel, goods and services in 15 countries. Based in Chicago, Groupon was launched there in November 2008, launching soon after in Boston, New York City and Toronto.


text:
descartewasamagician comments on MRW a gif takes too long to loadjump to contentmy subredditsannouncementsArtAskRedditaskscienceawwblogbookscreepydataisbeautifulDIYDocumentariesEarthPornexplainlikeimfiveFitnessfoodfunnyFuturologygadgetsgamingGetMotivatedgifshistoryIAmAInternetIsBeautifulJokesLifeProTipslistentothismildlyinterestingmoviesMusicnewsnosleepnottheonionOldSchoolCoolpersonalfinancephilosophyphotoshopbattlespicsscienceShowerthoughtsspacesportstelevisiontifutodayilearnedTwoXChromosomesUpliftingNewsvideosworldnewsWritingPromptsedit subscriptionsfront-all-random | tifu-philosophy-science-explainlikeimfive-television-gadgets-funny-Jokes-news-pics-food-TwoXChromosomes-photoshopbattles-aww-videos-Documentaries-Art-listentothis-WritingPrompts-books-UpliftingNews-dataisbeautiful-askscience-Futurology-creepy-EarthPorn-todayilearned-movies-history-gaming-nosleep-OldSchoolCool-mildlyinteresting-Music-Showerthoughts-GetMotivated-nottheonion-personalfinance-InternetIsBeautiful-DIY-sports-gifs-Fitness-space-IAmA-AskReddit-LifeProTips-worldnewsmore » reactiongifscommentsrelatedother discussions (3)want to join? login or register in seconds|Englishlimit my search to /r/reactiongifsuse the following search parameters to narrow your results:subreddit:subredditfind submissions in "subreddit"author:usernamefind submissions by "username"site:example.comfind submissions from "example.com"url:textsearch for "text" in urlselftext:textsearch for "text" in self post contentsself:yes (or self:no)include (or exclude) self postsnsfw:yes (or nsfw:no)include (or exclude) results marked as NSFWe.g. subreddit:aww site:imgur.com dogsee the search faq for details.advanced search: by author, subreddit...this post was submitted on 09 Mar 20143,068 points (95% upvoted)shortlink: remember mereset passwordloginSubmit a new .gifreactiongifssubscribeunsubscribe409,541 readers689 users here now
created by [deleted]a community for 2 yearsmessage the moderatorsMODERATORSkjoneslolsoupyhandsTheLegitMidgitjaxspiderHandicapperGeneralRswanykreiusAutoModeratorTheSonarManWithoutModem...and 11 more »306730683069MRW a gif takes too long to load (i.imgur.com)submitted 5 months ago by kamStock8596 commentssharecancelloading...sorted by: bestbesttopnewhotcontroversialoldrandomyou are viewing a single comment's thread.view the rest of the comments →[–]descartewasamagician 1 point2 points3 points 5 months ago (0 children)first world problems permalinkaboutblogaboutteamsource codeadvertisejobshelpwikiFAQreddiquetterulescontact ustoolsmobilefirefox extensionchrome extensionbuttonswidget<3reddit goldstoreredditgiftsreddit.tvradio redditUse of this site constitutes acceptance of our User Agreement {Genitive} and Privacy Policy {Genitive}. © 2014 reddit inc. All rights reserved.REDDIT and the ALIEN Logo are registered trademarks of reddit inc.close this windowyou'll need to login or register to do thatcreate a new accountall it takes is a username and passwordprivacy philosophy
username:try anotheravailable!account recovery email: (optional)we only send email at your requestpassword:verify password:remember mecreate accountis it really that easy? only one way to find out...loginalready have an account and just want to login?username:password:remember merecover passwordloginclose this windowinterface languageEnglish [en]العربية [ar]Беларуская мова [be]български език [bg]Bosanski [bs]català [ca]česky [cs]dansk [da]Deutsch [de]Ελληνικά [el]English (Australia) [en-au]English (Canadian) [en-ca]English (Great Britain) [en-gb]English [en-us]Esperanto [eo]español [es]español [es-ar]eesti keel [et]Euskara [eu]فارسی [fa]suomi [fi]français [fr]Gàidhlig [gd]עברית [he]मानक हिन्दी [hi]hrvatski [hr]Magyar [hu]Հայերեն լեզու [hy]Bahasa Indonesia [id]íslenska [is] (*)italiano (Italy) [it]日本語 [ja]ಕನ್ನಡ [kn_IN]한국어 [ko]Latin [la]1337 [leet]LOL [lol]lietuvių kalba [lt]latviešu valoda [lv]Nederlands [nl]Nynorsk [nn]Norsk [no]Arrrrrrrr! [pir]polski [pl]português [pt] (*)português [pt-pt]português brasileiro [pt_BR]română [ro]русский [ru]slovenčina [sk]slovenščina [sl]српски језик [sr]Srpski [sr-la]Svenska [sv]தமிழ் [ta]ภาษาไทย [th]Türkçe [tr]українська мова [uk]Tiếng Việt [vi]中文 [zh] (*) incomplete volunteer to translateπ Rendered by PID 17731 on app-165 at 2014-08-22 06:03:50.333972+00:00 running b5552ef country code: US.

value: Reddit (, stylized as reddit) is an American social news aggregation, web content rating, and discussion website. Registered members submit content to the site such as links, text posts, images, and videos, which are then voted up or down by other members.


text:
Something Beautiful by Tony Lindsay on Amazon Music - Amazon.com
MP3 Music, November 1, 2018
Audio CD, November 1, 2018
$20.88 $33.06
$20.88 1 Used from $33.06 4 New from $20.88
Copyright: ℗© 2018 Tony Lindsay
ASIN: B07K2Q6XCB
Amazon Best Sellers Rank: #262,038 Paid in Albums (See Top 100 Paid in Albums)
#383 in Contemporary R&B (Albums)
Andrew R. Ebon
Tony Lindsay is smooth as silk.

value: Amazon.com, Inc. ( AM-ə-zon) is an American multinational conglomerate which focuses on e-commerce, cloud computing, digital streaming, and artificial intelligence.


text:
giving: the pot-smoking beggar | aliens and strangers
← prophets for hire: preaching God in a congregation’s image
giving: the righteous man →
January 13, 2010 · 10:24 am
Everyday he stands in front of the Double Red Rose, one of three small grocers in town. His head is too big for his body, and his legs are twisted and misshapen. He limps slowly as he approaches those who enter the store. He speaks quietly — I’m not sure if he’s timid or embarrassed, shy or afraid. He’s never asked me for money, but every time I see him he whispers his request for food. One of the clerks at Double Red Rose tells me he’s homeless and usually sleeps in front of the shop. I ask if many people help him; the answer is “not many.” The ladies next door are cooking in the dirt over charcoal in front of their “restaurant.” They tell me the boy’s dad has never been around, and his mom passed away some time ago. They also report that he smokes pot when he has an extra hundred shillings (about eight cents), and they point to where he buys it. His name is Steve. He can’t be a year over 14, and he’s just one of the beggars I see and know in Geita.
When I lived in downtown Nashville, I was often asked for money. My roommates and I devised a set of “guidelines.” In short, we were not, under any circumstance, to give money — but food, or a ride, or even a bus ticket was okay. We were never to drive anyone anywhere alone. I often found myself explaining that I couldn’t give money, but would be happy to buy a meal. Some complained that I didn’t trust them. Others changed — in the middle of our conversation — the reason for their request from “I’m hungry” to “I need to buy diapers for my family.” I’d offer to buy the diapers, as they walked away grumbling. There were some who seemed grateful for the offer of a meal, and I’d agree to meet them in ten minutes with food. When I’d return, having bought a combo meal from McDonalds, they’d be nowhere in sight. But there were always a few who waited patiently for my return, or accompanied us to a restaurant — and were no longer hungry… for that one day.
I’ve read that some of the guys with signs on interstate exit ramps make over $300 a day, “working” less than eight hours.
I’ve been approached by the same guy in the same gas station, telling the same sad story, two weeks after he yelled at me for offering food in place of money.
I’ve had kids in China cling to my leg with one hand while holding their plastic bowl for coins in the other. And I knew their pimp was watching the performance from only a few meters away. I also knew the children wouldn’t receive any money I gave. I offered food, and they refused to eat, maybe knowing they’d be beaten if they did?
More people ask me for money in Tanzania than anywhere else I’ve been. There is Steve, but he is only one of many. My electrician asks me for extra money; so does the guy doing my masonry work. My friends ask me for loans that I know will never be repaid. The bicycle taxi guys ask for sodas, as do the vendors who sell sodas. Teenagers come by the house with petitions, trying to raise enough money for their school fees. The only English many children know is, “Give me my money.” Even my landlord, on a day he was frustrated with my decisions concerning the house, asked me for 20,000 shillings for his trouble, so that he and some friends could go out to dinner.
And Jesus says, “Give to everyone who asks you.”
I have struggled a lot with giving and generosity. And I still struggle. Christie and I have agreed if we ever cease to struggle with this issue, we will promptly remove ourselves from the mission field. But we have limited funds to share with others. And we know many don’t actually need the help for which they’re asking. They would use our money to buy alcohol or pot. Most will ask us for money again the next day, whether we give today or not. Sometimes a beggar will walk past a dozen Tanzanians, asking them for nothing — in order to ask us for money. And some things we’re asked for are not within our power, ability, or pocketbooks to give — such as passage to the U.S., a full year’s worth of school fees, or our truck.
But Jesus says, “Give to everyone who asks you.”
I don’t have all the answers to these problems. But, as shallow as my feet are planted in the soil of my decisions, and as uncomfortable as it makes me, I’ve attempted to determine a place to stand concerning these issues. Over the next week or so, I intend to “publish” my conclusions, though I view them more as starting blocks than finish lines. I will post many of my thoughts in the form of 3-column Bible studies, because I believe the process through which I’ve come is at least as meaningful as the decisions it has prompted.
If you care to join me in these studies, the first passage will be Psalm 112.
Tagged as beggars, generosity, giving
8 responses to “giving: the pot-smoking beggar”
Looking forward to reading what you are/have learned. I do think that the more things go on the more “needy” people we will see. makes it hard because you know there are legitimate needs but the shysters make it difficult to feel empathy.
one of the major questions i’ve had to ask during my studies has been:
does Christ seem concerned with whether or not the need is legitimate?
Pingback: giving: the righteous man « aliens and strangers
Pingback: giving: the farmer and his harvest « aliens and strangers
Pingback: giving: blow the trumpets and give me my reward « aliens and strangers
Pingback: missionary predicament: machine gun bribery | aliens and strangers

value: WordPress (WP, WordPress.org) is a free and open-source content management system (CMS) written in PHP and paired with a MySQL or MariaDB database. Features include a plugin architecture and a template system, referred to within WordPress as Themes.