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WO1991005571A1 - Procede de fabrication des facteurs de croissance des cellules de la moelle osseuse - Google Patents

Procede de fabrication des facteurs de croissance des cellules de la moelle osseuse Download PDF

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WO1991005571A1
WO1991005571A1 PCT/US1990/005686 US9005686W WO9105571A1 WO 1991005571 A1 WO1991005571 A1 WO 1991005571A1 US 9005686 W US9005686 W US 9005686W WO 9105571 A1 WO9105571 A1 WO 9105571A1
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Prior art keywords
bone marrow
culture
dithiocarbamate
ddtc
factors
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Richard F. Borch
Therese K. Schmalbach
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University of Rochester
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University of Rochester
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Priority claimed from US07/418,549 external-priority patent/US5035878A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0669—Bone marrow stromal cells; Whole bone marrow
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • dithiocarbamates and their dimers are clinically useful compounds of relatively low toxicity toward mammals.
  • Various sulfur- containing compounds including NaDDTC have been suggested as immunostimulant medicines. See U.S. Patent No. 4,148,885 (Renoux et al.), issued April 10, 1979.
  • dithiocarbamates or their dimers have been used to inhibit the undesirable side effects of platinum compounds such as the square planer platinum (II) complexes used as antineoplastic agents. See U.S. Patent Nos.
  • platinum compounds useful as antineoplastic agents are not limited to platinum (II) compounds, because it has been found that platinum (IV) compounds can be administered in much the same manner as platinum (II) compounds, apparently because these six- ligand complexes break down in vivo to square planar complexes of the platinum (II) type.
  • No. 4,426,372 has been shown to be effective in clinical trials. That is, this method substantially reduces the side effects of platinum-containing drugs. These side effects include both kidney toxicity and bone marrow toxicity.
  • the amount of dithiocarbamic "rescue agent" is likely to be in the range of 100 mg/kg to 400 mg/kg (intravenously) and can range as high as 750 mg/kg (intraperitoneally), also in mice.
  • a dosage of less than 50 mg/kg of body weight of dithiocarbamate is not likely to be fully effective in providing relief from or prevention of kidney damage.
  • myelosuppres- sion As noted previously, much less is known about treatments for bone marrow toxicity. Some anti-cancer drugs, both platinum-containing and platinum-free, can seriously damage the blood-forming function of the bone marrow—an effect sometimes referred to as myelosuppres- sion.
  • drugs causing significant myelosuppres- sion effects are cytotoxic antibiotics and antibiotic derivatives, other cytotoxic drugs, antimetabolites (which inhibit processes involved in DNA formation) , alkaloid-type anti-tumor agents, alkylating agents, and heavy metal complexes (particularly Pt complexes such as "Carboplatin”) .
  • a method to produce protective factors to counteract or to protect against the side effects of these drugs would be a highly welcome addition to the field of cancer treatment.
  • the present invention provides a process for obtaining one or more bone marrow cell growth factors having granulocyte/macrophage progenitor cell colony stimulating activity, said process comprising: (a) adding to the culture medium in an in vitro, established bone marrow culture a growth factor- stimulating amount of a dithiocarbamate compound of the formula (I) :
  • about 0.03-1.0 mmoles/1 of medium of the dithiocarbamate will be employed to stimulate the production of growth factor(s), most preferably about 0.03-0.2 mmole/1 of culture medium.
  • the present invention also encompasses novel bone marrow cell growth factors produced by the present process, as well as pharmaceutical dosage forms of said factors in a pharmaceutically acceptable medium.
  • the present invention further comprises a method for the in vivo stimulation of one or more bone marrow cell growth factors, e.g., to treat damage to, or to stimulate, the blood-forming function of the bone marrow of a living mammal by administering an effective amount of one or more of said pharmaceutical dosage forms, or by administering an effective amount of a dithiocarbamate of formula I.
  • Dithiocarbamic compounds of the formula I are surprisingly effective for the stimulation of, or for the treatment of damage to, the blood-forming function of the bone marrow of a living mammal when administered in doses which preferably do not cause the panic response described previously.
  • This discovery is disclosed and claimed, for example, in parent application Serial No. 07/418,549 and in Borch et al. (U.S. Patent No. 4,938,949), the disclosures of which are incorporated by reference herein.
  • Amounts of dithiocarbamic compounds in excess of 30 mg/kg of body weight of small mammals are not needed in this invention, and would be very excessive for large mammals such as humans particularly in the case of damage caused by anticancer drugs, e.g., platinum- containing drugs. At least some beneficial response to the dithiocarbamic compound is observable even in small mammals at dosage levels in the microgram/kg range.
  • a suitable dosage unit according to this invention can be in the range of about 0.001 to 30 mg per kilogram of body weight of the mammal, more preferably above about 0.003 and up to about 10 mg/kg of body weight.
  • the preferred dosage units for treating the side effects of these platinum-free drugs, and for stimulating the growth of bone marrow cells which have been damaged or compromised by other treatments or pathologies are the same as for the platinum-containing drugs, but dosages up to 300 mg/kg in mice (up to 75 mg/kg in humans) appear to be tolerated when adequate precautions are utilized, e.g. , sedation.
  • mg/m 2 dosing is equivalent in all species, including both large and small mammals. It has also been found that the gap between a suitable mg/kg dosage unit for a small mammal and a suitable mg/kg dosage unit for a human is somewhat less than might have been predicted by a skilled pharmacologist.
  • the therapeutic method of this invention applies primarily to mammals who have already suffered some myelosuppression effects.
  • the dithiocarbamic compound more or less simultaneously with the myelosuppression-causing agent (i.e., the drug or radiation or the like).
  • the dithiocarbamic compound will be administered prior to, and continued after the myelosuppression-causing agent has been given to the patient.
  • the preferred dithiocarbamic compounds used in this invention are those of the aforementioned formula (I), R ⁇ R 2 NCSSM, wherein M is a pharmaceutically acceptable cation, and R x and R 2 are lower aliphatic or hydroxy-substituted lower aliphatic groups (e.g., a polyhydroxy-substituted lower C 3 -C 6 -alkyl group or a (C 2 - C 6 )-alkyl).
  • the preferred route of administration of these compounds is parenteral, e.g., intravenous, and a suitable unit dosage can be dissolved, suspended, or otherwise combined with a pharmaceutically acceptable carrier such as an aqueous medium.
  • a pharmaceutically acceptable carrier such as an aqueous medium.
  • the dimers e.g., disulfiram
  • the preferred route of administration is oral.
  • the method and compounds of this invention can find application whenever the blood-forming function of the bone marrow of a living mammal has been damaged or inhibited.
  • the present treatment method can be used to stimulate the production of blood-forming bone marrow cells in the case of a bone-marrow transplant recipient, most of, or all of whose autologous bone marrow cells have been destroyed prior to marrow transplantation. Stimulation of the blood-forming bone marrow cells is also desirable in the treatment of diseases such as hypoplastic anemia.
  • clinical use of the method and dosage units of this invention can be carried out in combination with known antitumor agents and can be more or less simultaneous with (or even previous to) the administration of the antitumor agent, although typically the antitumor agent will be administered first. It is generally desirable that, when the antitumor agent is administered first, the dithiocarbamate is given to the treated mammal within three hours.
  • Myelosuppression (toxicity to the blood-forming cells of the bone marrow) is a serious and frequently dose-limiting side effect of most cancer drugs used in the cancer clinic today. Because these are rapidly dividing cells, they are particularly susceptible to the toxic effects of the drug used to control diseases of cell proliferation.
  • the stem cell is the most primitive of the bone marrow cells; it represents less than 0.1% of the cells of the marrow, yet it is capable of differentiating to produce progenitor cells for all of the blood cell lines (red cells, lymphocytes, granulocytes, and platelet precursors) .
  • the stem cell is also a self-replenishing cell in that it can undergo division to generate additional stem cells.
  • stem cells have been only recently specifically isolated and characterized, and then only in mice, an estimate of _ their numbers can be obtained using the spleen colony assay (CFU-S) . Maintenance of an appropriate population of stem cells is obviously critical to survival of mammals and perhaps other organisms.
  • the granulocyte precursor is one of the most important and frequently damaged progenitor cell in the bone marrow. Its clinical importance lies in the role that the granulocyte plays in fighting infections. Patients with markedly reduced granulocyte counts resulting from cancer chemotherapy are highly susceptible to infection from a variety of organisms and, if bone marrow function does not recover quickly enough, they can succumb to infection rather than the primary malignancy for which they have been receiving treatment.
  • the granulocyte precursor derives from differentiation of a stem cell; this precursor can undergo subsequent amplification and differentiation to produce a mature granulocyte.
  • the granulocyte precursor is more abundant in the marrow than the stem cell, and its numbers can be estimated using the CFU-GM assay.
  • dithiocarbamates selectively stimulate proliferation of cultured bone marrow cells in vitro.
  • dithiocarbamates such as _ sodium diethyldithiocarbamate (DDTC) do not alter the number of bone marrow cells proliferating in vivo in the absence of myelotoxic insult.
  • DDTC sodium diethyldithiocarbamate
  • antineoplastic agents and treatment techniques are a particularly important cause of myelosuppression.
  • These antineoplastic treatments fall into two broad categories: radiation therapy and drugs.
  • the drugs which have adverse effects upon blood formation e.g., bone marrow toxicity
  • fall into several categories including cytotoxic antibiotics isolated from cultures of various species of Streptomyces and derivatives of such antibiotics (bleomycin, daunorubicin, dactinomycin, doxorubicin hydrochloride) , other cytotoxic agents which are not necessarily antibiotic derivatives, antimetabolites such as 5- fluorouracil, cytarabine, and thioguanine; alkaloid-type compounds including alkaloids extracted from natural sources such as the periwinkle plant and similar herbs (vincristine sulfate, vinblastine sulfate) , DNA synthesis inhibitors and DNA crosslinkers which can be, for example, alkylating agents such as thiotepa or busul
  • an antibiotic derivative of a 2-chloroethyl- 5 containing compound or a cytotoxic agent can be a DNA synthesis inhibitor and/or an alkylating agent.
  • the mode of action of an antineoplastic drug is unknown, e.g., in the case of dacarbazine.
  • Adriamycin Doxorubicin hydrochloride
  • Streptomyces-produced antibiotic derivative which is known to cause bone marrow suppression effects, _ primarily of leukocytes.
  • careful hematologic monitoring is required when this drug is being administered to produce regression in neoplastic conditions.
  • nitrosourea typically have the following formula:
  • R* is an organic group such as an aliphatic or cycloaliphatic radical or a second 2-chloroethyl group.
  • R* is an organic group such as an aliphatic or cycloaliphatic radical or a second 2-chloroethyl group.
  • R* is an organic group such as an aliphatic or cycloaliphatic radical or a second 2-chloroethyl group.
  • One widely used compound of this type is l-3-bis(2- chloroethyl)-l-nitrosourea, also known as BCNU or BiCNU or carmustine.
  • the bis-(2-chloroethyl)-amino functional group is particularly common, e.g., as in chlorambucil or cyclophosphamide.
  • This bis-substituted group has the formula (C1CH 2 CH 2 ) 2 N- and can be substituted directly on an aliphatic chain or an aromatic or cycloaliphatic or heterocycloaliphatic ring (or indirectly, whereby N is part of a carbamate linkage or the like) .
  • the so-called "nitrogen mustard” derivatives typically contain the bis-(2-chloroethyl)- amino group and can be highly toxic if not carefully administered.
  • the nitrogen-containing platinum monodentates and bidentates myelosuppression can occur when the ligands include ammonia, diaminocyclohexane and its derivatives, alkylene and diamines (e.g., ethylenediamine) , alkyl-substituted amines, C 3 - and C 5 - cycloalkyl amines, and the like.
  • the ligands include ammonia, diaminocyclohexane and its derivatives, alkylene and diamines (e.g., ethylenediamine) , alkyl-substituted amines, C 3 - and C 5 - cycloalkyl amines, and the like.
  • Suitably selected tetravalent Pt complexes can behave like Pt(II) complexes after administration to a living organism. Removal of axial ligands in vivo accounts for the Pt(II)-like activity, at least to some extent.
  • a particular preferred species of Pt(IV) complex is chlorohydroxy-isopropylamineplatinum ("CHIP").
  • CHIP chlorohydroxy-isopropylamineplatinum
  • “CHIP” 11 like other "second-generation” platinum-containing therapeutic agents is low in kidney toxicity compared to the "first generation” agents but, unfortunately, is high in bone marrow toxicity.
  • Various Pt(II) compounds of demonstrated anti ⁇ tumor utility, e.g., "TNO-6" and "CBDCA” see U.S. Patent No. 4,137,248) also showed increased bone marrow toxicity.
  • Pt(II) compounds can be characterized by the formula: (R'NH 2 ) (R"NH 2 )Pt(X 1 ) (X 2 ) where X 1 and X 2 are the same or different and are halogen, OH, water, carboxyl, sulfato, or sulfate, or, taken together, the residue of a polycarboxylic acid; X 1 and X 2 preferably are S0 3 H or -C0 2 -, particularly as the residue of a polycarboxylic acid such as 1,1- cyclobutane-dicarboxylic acid, trimellitic acid, etc; R' and R" are the same or different and are halogen or an aliphatic group, or taken together, the aliphatic residue of a heterocyclic moiety which includes both N- atoms.
  • dithiocarbamic compounds or dithiocarbamates as used in this application is intended to refer to compounds containing the functional group R ⁇ N-CS-S-, wherein R_ and R 2 are the same or different and represent different aliphatic or cycloaliphatic or heterocycloaliphatic groups, e.g., (C x -C 6 )alkyl, (C 5 - C 10 )cycloalkyl or five- to ten-membered heterocyclic groups, unsubstituted or substituted by hydroxyl.
  • One of the two groups, R : and R 2 can be hydrogen.
  • R x and R 2 taken together with the N-atom, can be a 5- or 6-member N-heterocyclic ring which is aliphatic or aliphatic interrupted by a ring oxygen or a second ring nitrogen.
  • the dangling valence bond is linked to a group of the formula -S-CS-NR 3 R ⁇ , wherein R 3 and R are defined in the same manner as R : and R 2 .
  • the cation can be of the ammonium-type or can be derived form a monovalent or divalent metal such as an alkali or alkaline earth metal, cations which provide good water solubility and low toxicity being preferred, e.g., Na + , K + , Zn ++ and the like.
  • the group R ⁇ N-CS-S- is linked to a hydrogen atom which is ionizable, particularly at a pH above about 5. Since the dithiocarbamic acids are not very stable in vitro, it would appear to be only marginally operative, and not advantageous, to use the dithiocarbamic acid form of the myelosuppression treatment agents of this invention. However, these acids are generally soluble in polar organic solvents such as alcohol, and they would have some tendency to form stable alkali metal salts in body fluids.
  • Dithiocarbamates and related compounds have been reviewed extensively in a work by G.D. Thorn et al. entitled “The Dithiocarbamates and Related Compounds," Elsevier, New York, 1962. As explained in Chapter 2 of Thorn et al. , the preparation of dithiocarbamates is very simple.
  • the compounds of the formula R ⁇ NCSSH or R ⁇ NCSSNa can be formed by reaction of carbon disulfide with a secondary amine, typically in alcoholic or aqueous solution. The usual practice is to carry out this reaction in the presence of NaOH, so that the sodium dithiocarbamate salt is formed.
  • sodium dimethyl dithiocarbamate is formed from CS 2/ NaOH and dimethylamine.
  • N-methyl,N-ethyldithiocarbamates include: N-methyl,N-ethyldithiocarbamates, hexamethylenedithiocarbamic acid, sodium di(beta- hydroxyethyl) dithiocarbamate, various dipropyl, dibutyl and diamyl dithicarbamates, sodium N-methyl,N- cyclobutylmethyl dithiocarbamate, sodium N-allyl-N- cyclopropylmethyldithiocarbamate, cyclohexyla yl- dithiocarbamates, dibenzyl-dithiocarbamates, sodium dimethylene-dithiocarbamate, various pentamethylene dithiocarbamate salts, sodium pyrrolidine-N- carbodithioate, sodium piperidine-N-carbodithioate, sodium morpholine-N-car
  • R x of the structure R ⁇ N-CS-S- is a hydroxy-substituted or, preferably, a polyhydroxy-substituted lower alkyl group having up to 6 carbon atoms.
  • R_ can be HO- CH 2 -CHOH-CHOH-CHOH-CHOH-CH 2 -.
  • R 2 can be H or lower alkyl (unsubstituted or substituted with one or more hydroxyl groups).
  • Stearic problems can, of course, be minimized when R 2 is H, methyl, or ethyl.
  • a particularly preferred compound of this type is an N-methyl-glucamine dithiocarbamate salt, the most preferred cations of these salts being sodium or potassium.
  • lower refers to radicals having one to six carbon atoms. Water solubility and/or biocompatibility problems can be greatly increased when the number of carbon atoms exceeds six. Of the unsubstituted alkyl groups, the ethyl radical appears to provide a high level of water solubility coupled with relatively low toxicity. Nevertheless, compounds such as sodium diethyl- dithiocarbamate (NaDDTC) are not necessarily well tolerated by humans and other mammals (even smaller mammals) when administered at levels above 50 mg/kg of body weight.
  • NaDDTC sodium diethyl- dithiocarbamate
  • the dithiocarbamate derivative of N-methyl glucamine (e.g., sodium N-methyglucamine dithiocarbamate) was synthesized in 1984 and has been shown to inhibit the nephrotoxicity of the compound "cisplatin” (cis-dichlorodiammine platinum [II]). Moreover, the polyhydroxylated side chain appears to reduce somewhat the dithiocarbamate side effects described above.
  • dithiocarbamates include the alkali or alkaline earth metal salts wherein the anion is di-n-butyldithiocarbamate, di-n- propyldithiocarbamate, pentamethylenedithiocarbamate, and tetramethylene dithiocarbamate and those compounds wherein R : and/or R 2 of the formula R ⁇ N-CS-S- is a beta- hydroxyethyl.
  • Disulfiram is commercially available and has been used in the treatment of alcoholism to help the patient remain in a state of self-imposed sobriety. This treatment is carried out by oral administration of disulfiram in tablet form. Disulfiram has relatively low solubility in polar solvents, whereas diethyldithiocarbamate monomeric salts and hydroxy- substituted alkyl dithiocarbamate monomeric salts are _ highly soluble in water, e.g., in molar quantities, and are also soluble in alcohol.
  • parenteral modes of administration can be used, e.g., intramuscular injection or introduction through the intraperitoneal route.
  • Oral administration can also be employed to administer dithiocarbamates in accord with the present method.
  • the dosage units of this invention are most effective by the intravenous route.
  • mg/kg i.e., mg/kg of body weight
  • mg/kg per day mg/kg dosage unit
  • an average effective dose can vary from species to species, due to differences in metabolic rates.
  • Smaller mammals such as rats and mice metabolize drugs (convert the drugs to other compounds in vivo) more effectively than larger mammals such as dogs and humans.
  • Theoretical studies of drug metabolic rates in general tend to confirm that there is a rough inverse correlation between drug metabolic rate and the surface area of the body of the mammal.
  • a dosage expressed in mg/m 2 would be roughly equivalent in all species, regardless of body area, i.e., an ED 50 of 100 mg/m 2 in a human would also be 100 mg/m 2 in a mouse.
  • Dose in mg/m 2 Constant x dose in mg/kg.
  • the constant for human, dog, rat and mouse species are, respectively; 37, 20, 5.2, and 3.0. Expressed in relative terms, the human constant is almost twice the dog constant (1.9), the human constant is over 7 times the rat constant, and the human constant is 12.3 times J> the mouse constant.
  • the human dose of 5 NaDDTC can be as much as a sixth to a third, e.g., one- fourth of the dose for mice; hence, a dose in mice of, for example, 30 mg/kg works out in practice to be 5 to 10 mg/kg, most typically 7.5 mg/kg for humans.
  • a dosage of 0.3 mg/kg (1 mg/m 2 ) can 0 provide some useful effect in humans and has even been observed to show some bone marrow-restoring effect in mice.
  • a reliable effective dose range is, for example, about 1.0 to about 145 mg/m 2 , more preferably 130 mg/m 2 , regardless of species. For all species, the dosage of 5 130 mg/m 2 is ample and may be unnecessarily large.
  • Suitable dosage units can be less than 90 mg/m 2 or, if desired, less than 75 mg/m 2 .
  • dosage units in mg/kg are best calculated by dividing the mg/kg dose for mice by about 4 (instead of by 12.3). Accordingly, a 0 dose for mice of, say, 30 mg/kg would work out to about 7.5 mg/kg in a human, and a dose for mice of 10 mg/kg would work out to about 2.5 mg/kg in a human.
  • dithiocarbamic treatment agents of this invention 5 exhibit a rather typical sigmoidal logarithmic dose- response curve, but the placement of this curve with respect to the dose and response axes is surprising.
  • the percent of surviving stem cells in the test animals is indicated by the ordinate, and the dosage is indicated in 10-fold intervals (log 10 dose units) with respect to the abscissa.
  • the resulting plot shows that optimal bone marrow protection can be obtained at dosages well below 50 mg/kg of body weight, and even at well below 30 mg/kg.
  • a response can be observed at extremely low dosages (above sub-microgram/kg levels but still below 3 ⁇ g/kg, e.g., about 1 ⁇ g/kg), and significant protection appears to be obtained, even in mice, at dosages as low as 3 ⁇ g/kg, i.e., 0.003 mg/kg.
  • Dosages approaching 30 mg/kg (even in mice) appear to be unnecessarily high in the context of the method of this invention, hence a preferred range for a dosage unit of this invention is about 0.3 to 10 mg/kg of body weight of the mammal.
  • the "flat" portion of the sigmoidal curve appears to be reached at dosages as low as 0.3 mg/kg, but it can be desirable to exceed this dosage level in order to provide assurance that efficacy will be high.
  • a particularly preferred upper limit for the human dose appears to be about 10 mg/kg, more preferably 3.0 or even 2.5 mg/kg.
  • a useful range is, for example, 1-200 mg/m 2 , more prefer- ably about 1-75 mg/m 2 , as explained previously.
  • a particularly preferred form of a dosage unit of this invention is obtained by dissolving a dithiocarbamate salt in an aqueous medium (e.g., normal saline) , measuring out a dosage unit in the range of 0.001 to 30 mg per kilogram of body weight of the mammal to be treated, and sealing the resulting dosage unit in a vial (e.g., a glass or plastic vial) adapted for use in a conventional intravenous administration technique.
  • the dosage unit can be dissolved in a conventional plastic intravenous drip bag, in which case the dosage unit can be diluted with an aqueous solution of a typical intravenous administration fluid. (The potential chelating or complexing effects of the dithiocarbamic compound should be taken into account, with respect to such fluids. )
  • a dosage unit of the _ dithiocarbamic compound can be extended with a standard solid pharmaceutically acceptable extender (e.g., mannitol) and packaged in dosage unit form for solution later on in a fluid suitable for intravenous administration.
  • a standard solid pharmaceutically acceptable extender e.g., mannitol
  • Adjuvants, excipients, and the like can be included.
  • a particularly preferred unit dosage of this invention comprises about 0.01 to about 10 mg/kg of the dithiocarbamic myelosuppression treatment agent, the treatment agent being dissolved in a liquid pharmaceutically acceptable carrier comprising an aqueous medium.
  • suitable pharmaceutically acceptable carriers are available to those skilled in the art.
  • BDFi mice were used and the drugs were ad ⁇ ministered by intravenous (iv) injection in the tail vein.
  • Sodium diethyldithiocarbamate (DDTC) was administered at various dosages 3 hours after administration of an anticancer drug.
  • Bone marrow cells were harvested 24 hours after anticancer drug treatment (21 hours after NaDDTC) .
  • Toxicity to stem cells was 0 evaluated using the spleen colony (CFU-S) assay; toxicity to granulocyte progenitors was evaluated using an in vitro clonogenic (CFU-GM) assay.
  • mice were randomly divided into four groups of four animals each; one group served as a no- 5 treatment control, one group received DDTC alone (the "DDTC group”), one group received anticancer drug alone (the “drug-only group”), and one group received anticancer drug followed by DDTC 3 hours later (the "drug and DDTC group”). Twenty-four hours after drug treatment, the mice were killed by cervical dislocation, the femurs were removed, and the marrow cells were flushed out of the bone and counted.
  • mice For the CFU-S assay, 5-15 x 10 4 cells were injected via the tail vein into recipient mice that had just received a bone marrow lethal dose of radiation. Twelve days after injection of donor marrow cells, the mice were killed by cervical dislocation, the spleens were removed, and the colonies of cells growing on the surface of the spleen were counted. The data are normalized to represent the number of colonies formed/10 5 cells injected and are reported as the percent of colonies formed compared to the control group.
  • the data obtained from the DDTC group and the no-treatment group tends to confirm that DDTC has little or no stimulant effect upon healthy bone marrow in vivo. That is, DDTC has negligible effects on the stem cell and granulocyte precursor populations in normal mouse bone marrow.
  • the colony counts for the DDTC group were within 10% of no-treatment group values for both CFU-S and CFU-GM in all cases.
  • dose-dependent toxicity toward both CFU-S and CFU-GM was observed for carmustine (BCNU) and adriamycin.
  • BCNU carmustine
  • DDTC provided significant protection against BCNU toxicity to both stem cells and granulocyte progenitors at all doses of BCNU tested.
  • adriamycin reduction of toxicity was observed at all doses but was less impressive at the highest adriamycin dose tested.
  • mitomycin an anticancer drug of the antibiotic type
  • mitomycin an anticancer drug of the antibiotic type
  • Cis-diammine(eyelobutanedi- carboxylato)platinum (II), or "CBDCA” was obtained from Johnson-Matthey, Inc. (Malvern, PA).
  • Sodium diethyl- dithiocarbamate (DDTC) was obtained from Sigma Chemical Company (St. Louis, MO).
  • Fischer's medium, ⁇ -MEM, L- glutamine, pokeweed mitogen, sodium bicarbonate (7.5% solution) , gentamicin, penicillin/streptomycin solution, and antibiotic/antimycotic solution were purchased from GIBCO (Grand Island, NY) .
  • Horse serum and fetal bovine serum were purchased from Hyclone Laboratories (Logan, UT) .
  • Salmonella typhosa lipopolysaccharide B was purchased from Difco Laboratories (Detroit, MI) . Methylcellulose (4A premium grade) was provided by Dow Chemical Company (Midland, MI). Falcon Petri plates and microscope slides were obtained from Fisher Scientific Company (Springfield, NJ) . All other plastic culture supplies and test tubes were obtained from VWR (Rochester, NY) .
  • mice Male 6- to 8-week-old C57BL/6J x DBA/2J mice were obtained from The Jackson Laboratories (Bar Harbor, ME) . Mice were housed 10/cage in plastic cages and allowed food and water ad libitum. All mice were acclimated for at least 7 days; the animals were then killed by cervical dislocation and both femurs and tibias were harvested for these experiments.
  • LTBMC Murine Loner-term Bone Marrow Cultures
  • the cultures were maintained in a fully humidified incubator, 5% C0 2 atmosphere, at 33°C. Weekly feeding was performed by replacement of the spent medium and nonadherent cells with 10 ml of fresh medium. Where specified, the medium also contained 10 "5 M hydrocortisone sodium hemisuccinate, to facilitate development and maintenance of the adherent cells.
  • GM-CFC Granulocyte/Macrophaqe Progenitor Cell
  • the morphology of the cells in the colony was verified by removing the colonies from the media with a finely drawn pipet, resuspending the colony in 0.4 ml of media ( ⁇ -MEM or Fischer's) supplemented with 1-5% serum (horse or FBS), spinning the colony onto a slide with a Cytospin centrifuge (500 rpm for 5 min.), and staining with Wright-Giemsa stain. Positive (maximally stimulating amounts of PWM-SCCM included in the culture medium) and negative (no mitogen added) controls were included with each assay. The formation of colonies under these conditions was indicative of colony-stimulating activity in the LTBMC supernatants.
  • CSA Colony Stimulating Activity
  • the cultures were allowed to grow for 5 to 6 weeks prior to experimentation. Twelve cultures were randomly divided into four groups (control, DDTC, CBDCA, and CBDCA followed by DDTC), 3 cultures per group. Drug solutions were prepared immediately prior to use with unsupplemented medium and filter sterilized. CBDCA (300 ⁇ M in 10 ml medium) was applied to CBDCA- and CBDCA/DDTC-treated groups while the control and DDTC- treated groups received medium only. Cultures were replaced in the incubator for one hour.
  • the medium/drug solution was then removed and DDTC (300 ⁇ M in 10 ml medium) was added to DDTC and CBDCA/DDTC groups, while the control and CBDCA cultures received medium only. After one hour, these solutions were removed, and 10 ml of supplemented Fischer's medium were added to each culture. At the specified time, this medium, along with any nonadherent cell groups, was removed, and the cells were pelleted by centrifugation (800 x g for 5 min.). The supernatants were subsequently evaluated for colony- stimulating activity in the GM-CFC assay as described above.
  • Salmonella typhosa lipopolysaccharide B (5 ⁇ g/ml) was added in place of drug to the supplemented Fischer's medium following drug treatment. At the specified times, the medium was removed and tested for colony-stimulating activity as described above.
  • the CSA production stimulated by various doses of DDTC was determined by treating triplicate cultures with the specified concentration of DDTC or media alone for one hour. This solution was then replaced with supplemented Fischer's medium. Forty-eight hours later, the medium was removed, non-adherent cells were pelleted by centrifugation, and the supernatant was tested for colony-stimulating activity.
  • Results are ratio of colonies/10 5 cells using supernatants from treated LTBMC compared to control LTBMC treated with growth medium alone, Mean ⁇ SEM from three experiments at each time point.
  • CSA was augmented almost 4-fold in supernatants from DDTC-treated cultures, and this level represented about 50% of the maximal stimulation observed with conditioned medium (PWM-SCCM) .
  • CBDCA had no significant effect on 5 CSA either alone or when added just prior to DDTC treatment.
  • DDTC enhanced CSA at concentrations from 100-1000 ⁇ M. These concentrations are readily achieved in the plasma of patients treated with DDTC, as demonstrated by R. Qazi et al., J. Nat. Cancer Inst., 10 .80, 1486 (1988).
  • EXAMPLE III To determine whether or not DDTC is enhancing production of a factor(s) that stimulates progenitor cells, two different agents known to have CSA were evaluated in combination with DDTC.
  • the hematopoietic microenvironment is believed to play a pivotal role in the regulation of blood cell production and differentiation.
  • Stromal cells are most likely responsible for elaborating the colony- stimulating factors that regulate the LTBMC system.
  • LTBMC containing stromal cells were established by the method of L.H. Williams et al., Exp. Hematol. , 16, 80 (1988). The cells growing in these cultures were plated in standard clonogenic assays, and no progenitor or stem cell growth was observed, thereby confirming the absence of hematopoietic progenitor cells.
  • DDTC modulates hematologic toxicity by inducing stromal cell production of a factor or factors that stimulate hematopoiesis.
  • DDTC stimulates proliferation of both stem cells and GM progenitors in vivo only after damage or inhibition of the blood-forming cells of the bone marrow has occurred, e.g., via pretreatment with a myelotoxic drug, CSA was increased by exposure to DDTC alone in vitro.
  • Treatment of LTBMC with a cytotoxic concentration of CBDCA had no effect on CSA, and CBDCA neither enhanced nor inhibited the DDTC response in vitro.
  • the response is presumably initiated by cytotoxic drug in vivo and by the addition of fresh medium in vitro.
  • the involvement of stromal cells in the DDTC response may also account for the variable results observed with different cytotoxic agents, because direct toxicity to stromal cells would be expected to reduce the DDTC response.
  • These results represent the first example of bone marrow proliferation resulting from induction of colony- stimulating factor(s) by a small molecule.
  • the identity of the factor(s) responsible for the CSA induced by DDTC is not known, several cytokines may be potential candidates.
  • GM-CSF and G- CSF may play a role in the DDTC response, but their effects may be secondary to release of another cytokine such as IL-l ⁇ , IL-1/3, IL-6, IL-3, or mixtures thereof.
  • Another cytokine such as IL-l ⁇ , IL-1/3, IL-6, IL-3, or mixtures thereof.
  • the concentration of tumor necrosis factor has also been found to be increased in culture.
  • the production of one or more growth factors can be accomplished in vitro by adding to the culture medium of an in vitro, established bone marrow culture a growth factor-stimulating amount of a previously described com ⁇ pound of the formula I (R : R 2 N(CS)SM) (preferably about 0.1 to about 1.0 millimole, e.g., about 0.2 to 0.5 millimole, of the compound per liter of culture medium) , separating the compound from the thus-treated culture, adding fresh culture medium to the thus-treated culture, and permitting the concentration of growth factor or factors to build up in the fresh medium.
  • a growth factor-stimulating amount of a previously described com ⁇ pound of the formula I (R : R 2 N(CS)SM) preferably about 0.1 to about 1.0 millimole, e.g., about 0.2 to 0.5 millimole, of the compound per liter of culture medium
  • This concentration appears to reach a peak in 8 to 72-96 hours (e.g., 24-48 hours) and then declines, because the growth factor or factors are continuously subject to consumption or utilization by the treated culture.
  • the growth factor or factors can then be isolated by removing the fresh medium from the treated bone marrow culture, and performing conventional steps used to con ⁇ centrate and purify cytokines.
  • this invention contemplates in vivo or in vitro stimulation of one or more bone marrow cell growth factors (having G/M cell CSA) via the exposure of bone marrow cells to small amounts of one or more of the previously-described dithiocarbamic compounds of the formula ⁇ NfCSJSM.
  • this invention can provide a surprisingly simple alternative to the use of cytokines such as the interleukins, and other highly complex cell growth stimulating factors which are difficult to synthesize in quantity without resorting to the use of genetically-engineered organisms. Therefore, the stimulation and proliferation of other cells which has been accomplished using IL-1 or IL-2 in the past, can be accomplished using thiocarbamates of formula I.
  • the stimu ⁇ lation and proliferation of LAK cells or of T-helper cell populations can also be accomplished in accord with the present invention.
  • the administration of DDTC or other dithiocarbamic compounds of the formula R 1 R 2 N(CS)SM for this purpose is particularly attractive in view of the low toxicity of these compounds, their high solubility in ordinary pharmaceutically acceptable media such as water, and their extraordinary efficacy in stimulating G/M cell CSA at very low doses.
  • Dosage units of this invention are ideal for time-intensive as opposed to time-diffusive use, i.e., essentially single-dose use.
  • the entire dose, undivided or divided into less that 5 or 10 increments, is administered over a very short period of time, e.g., less than 24 hours and preferably less than 8 hours (most preferably by a single injection) and preferably only in response to— and within 24 hours (preferably within 8 hours) of—an insult to the bone marrow (such as a radiation treatment or an anticancer treatment) .
  • This time-intensive use is easily distinguishable from continuous dosing and is particularly different from long-term regimens in which a compound is given repeatedly over a period of several days or weeks or in some other time-diffusive manner typically involving small doses.

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Abstract

On décrit un procédé servant à obtenir un ou plusieurs facteurs de croissance des cellules de la moelle osseuse, lesdits facteurs présentant une activité stimulatrice des colonies de cellules ascendantes des granulocytes et des monocytes. Ledit procédé consiste : (a) à ajouter au milieu de culture d'une culture in vitro et établie de moelle osseuse, une quantité suffisante de dithiocarbamate pour stimuler le facteur de croissance; (b) à séparer ledit dithiocarbamate de la culture de moelle osseuse traitée in vitro et à ajouter à celle-ci un nouveau milieu de culture, et à permettre à la concentration dudit/desdits facteur(s) d'augmenter dans ledit nouveau milieu de culture; et enfin (c) à isoler ledit/lesdits facteur(s) de croissance dudit nouveau milieu de culture.
PCT/US1990/005686 1989-10-10 1990-10-04 Procede de fabrication des facteurs de croissance des cellules de la moelle osseuse Ceased WO1991005571A1 (fr)

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US418,549 1989-10-10
US07/418,549 US5035878A (en) 1988-09-12 1989-10-10 Use of dithiocarbamates to counteract myelosuppression
US8322690A 1990-09-21 1990-09-21
US083,226 1990-09-21

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5638449A (en) * 1993-01-26 1997-06-10 Inria Institut National De Recherche En Informatique Et En Automatique Data transmission installation of the radio network type, and corresponding process
EP0809437A4 (fr) * 1995-02-17 2001-05-09 Medimmune Oncology Inc Thiols utilises pour favoriser la croissance de cellules hematopoietiques parentes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4148885A (en) * 1973-09-25 1979-04-10 Institut Merieux Immunostimulant medicine
US4581224A (en) * 1980-08-08 1986-04-08 Regents Of The University Of Minnesota Inhibition of undesired effect of platinum compounds
US4594238A (en) * 1980-08-08 1986-06-10 Regents Of University Of Minnesota Inhibition of undesired effect of platinum compounds
US4721096A (en) * 1986-04-18 1988-01-26 Marrow-Tech Incorporated Process for replicating bone marrow in vitro and using the same
US4938949A (en) * 1988-09-12 1990-07-03 University Of New York Treatment of damaged bone marrow and dosage units therefor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4148885A (en) * 1973-09-25 1979-04-10 Institut Merieux Immunostimulant medicine
US4581224A (en) * 1980-08-08 1986-04-08 Regents Of The University Of Minnesota Inhibition of undesired effect of platinum compounds
US4594238A (en) * 1980-08-08 1986-06-10 Regents Of University Of Minnesota Inhibition of undesired effect of platinum compounds
US4721096A (en) * 1986-04-18 1988-01-26 Marrow-Tech Incorporated Process for replicating bone marrow in vitro and using the same
US4938949A (en) * 1988-09-12 1990-07-03 University Of New York Treatment of damaged bone marrow and dosage units therefor

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
AACR ABSTRACTS, issued March 1984, Abstract No. 1471, GRINGERI et al., "The Effect of Diethyldithiocarbamate (DDTC) on Bone Marrow Cells Treated with Cis-Diaminedichloro Platinum (DDP) and its Analogs", page 371. *
ANNALS OF CLINICAL RESEARCH, Volume 3, issued 1971, SUNDERMAN, SR: "The Treatment of Acute Nickel Carbonyl Poisoning with Sodium Diethyldithiocarbamate", pages 182-185. *
BR. J. CANCER, Volume 59, issued 1989, PANNACCIULLI et al., "Effects of Diethyldithiocarbamate on Toxicity of Doxorubicin, Cyclophosphamide, and Cis-Diaminedichloroplatinum (II) on Mice Haematopoietic Progenitor Cells", pages 371-374. *
CANC. THEMOTHER. PHARMACOL., Volume 16, issued 1986, GAMELLI et al., "The Effect of Disulfiram of Cyclophosphamide-Mediated Myeloid Toxicity", pages 153-155. *
CANC. THEMOTHER. PHARMACOL., Volume 17, issued 1986, JONES et al., "Inhibition of Cis-Diaminedichloroplatinum (II) - Induced Renal Toxicity in the Rat", pages 38-42. *
CANCER RESEARCH, Volume 44, issued September 1984, EVANS et al., "Modification of Bone Marrow Toxicity to Cis-Diaminedichloroplatinum (II) in Mice by Diethyldithiocarbamate", pages 3686-3690. *
CANCER RESEARCH, Volume 46, issued June 1986, BODENNER et al., "Selective Protection Against Cis-Diaminedichloroplatinum (II) - Induced Toxicity in Kidney, Gut, and Bone Marrow by Diethyldithiocarbamate", pages 2751-2755. *
CANCER RESEARCH, Volume 48, issued 15 October 1988, GRINGERI et al., "Diethyldithiocarbamate Inhibition of Murine Bone Marrow Toxicity Caused by Diaminedichloroplatinum(11)...(11)", pages 5708-5712. *
CANCER RESEARCH, Volume 49, issued 01 December 1989, SCHMALBACH et al., "Diethyldithiocarbamate Modulation of Muring Bone Marrow Toxicity Induced by Cis-Diaminedichloroplatinum(II)", pages 6629-6623. *
FRESHNEY, R.I., "Culture of Animal Cells", Second edition, Published 1987, by ALAN R. LISS (N.Y.), pages 284-288. *
INTL. J. CELL CLONING, Volume 1, issued 1983, GORDON et al., "Spatial and Functional Relationship Between Human Hematopoietic and Marrow Stromal Cells In Vitro", pages 429-439. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5638449A (en) * 1993-01-26 1997-06-10 Inria Institut National De Recherche En Informatique Et En Automatique Data transmission installation of the radio network type, and corresponding process
EP0809437A4 (fr) * 1995-02-17 2001-05-09 Medimmune Oncology Inc Thiols utilises pour favoriser la croissance de cellules hematopoietiques parentes

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