Method for stimulating production of bone marrow cell growth factors using dithiocarbamates

A process is provided 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 of an in vitro, established bone marrow culture a growth factor-stimulating amount of a dithiocarbamate; (b) separating the said dithiocarbamate from the in vitro treated bone marrow culture, adding fresh culture medium to said in vitro treated bone marrow culture, and permitting the concentration of said growth factor or factors to increase in said fresh culture medium; and (c) isolating said growth factor or factors from said fresh culture medium.

BACKGROUND OF THE INVENTION 
At least as far back as the early 1970s, it was found that dithiocarbamates 
and their dimers (e.g., disulfiram) are clinically useful compounds of 
relatively low toxicity toward mammals. Various sulfur-containing 
compounds including sodium diethyldithiocarbamate (NaDDTC) have been 
suggested as immunostimulant medicines. See U.S. Pat. No. 4,148,885 
(Renoux et al.), issued Apr. 10, 1979. Also, 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. Pat. Nos. 4,426,372 (Jan. 17, 1984), 
4,594,238 (Jun. 10, 1986), and 4,581,224 (Apr. 8, 1986), all issued to R. 
F. Borch. The 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. 
The Borch method of, for example, U.S. Pat. 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. For 5 mg/kg of 
intravenously administered platinum compound in mice, 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. 
Although pharmaceutically acceptable dithiocarbamic compounds such as 
sodium diethyldithiocarbamate (NaDDTC) and disulfiram have relatively high 
LD.sub.50 values and are not considered highly toxic to mammals, there are 
scattered reports in the literature regarding strange behavior exhibited 
by rats or mice injected with NaDDTC. The true import of this literature 
became fully apparent during clinical trials of NaDDTC as a "rescue 
agent", i.e., as an agent for the reduction of side effects from the 
administration of platinum compounds. These clinical trials demonstrated 
that human patients given dosages of NaDDTC effective for "rescue" 
purposes (e.g., dosages on the order of 50-150 mg/kg of body weight) 
experienced extremely unpleasant effects which caused them to feel panic 
and discomfort. It was necessary to develop a technique of administration 
of the NaDDTC whereby the patient is sedated prior to receiving the 
dithiocarbamate. 
All available evidence indicates that the panic reaction to 
dithiocarbamates resulting from dosages of, for example, 50-150 mg/kg is 
not the result of any life-threatening process occurring in the body of 
the patient, nor is there any evidence of permanent or chronic effects or 
damage resulting from NaDDTC administration. After the course of 
dithiocarbamate administration has been completed, patients returned to 
normal and no sequellae of the panic reaction are observed. Moreover, it 
presently appears that some hydroxy-substituted analogs of NaDDTC may be 
even less toxic than NaDDTC itself. Nevertheless, further improvement in 
the treatment of toxic side effects of useful cytotoxic compounds is 
desirable. 
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 myelosuppression. Among the 
drugs causing significant myelosuppression 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. 
SUMMARY OF THE INVENTION 
The present invention provides a process for obtaining one or more bone 
marrow cell growth factors having granulocyte/macrophage progenitor cell 
colony stimulating activity (GM-CSA), 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): 
##STR1## 
wherein R.sup.1 and R.sup.2 are the same or different lower aliphatic or 
cycloaliphatic or heterocycloaliphatic groups, unsubstituted or 
substituted by hydroxyl, or one of R.sup.1 and R.sup.2, but not both, can 
be H, or R.sup.1 and R.sup.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 second ring nitrogen, and M is H or one 
equivalent of a pharmaceutically acceptable cation, in which case the rest 
of the molecule is negatively charged, 
or M is 
##STR2## 
wherein R.sup.3 and R.sup.4 are defined in the same manner as R.sup.1 and 
R.sup.2, thereby obtaining an in vitro treated bone marrow culture; 
(b) separating the said compound from the in vitro treated bone marrow 
culture, adding fresh culture medium to said in vitro treated bone marrow 
culture, and permitting the concentration of said growth factor or factors 
to build up in said fresh culture medium; 
(c) after an effective period of time, e.g., about 8 but less than 96 
hours, separating the said fresh culture medium from the in vitro treated 
bone marrow culture, thereby isolating said growth factor or factors from 
the in vitro treated bone marrow culture in essentially pure form. 
Preferably, about 0.03-1.0 mmoles/l of medium of the dithiocarbamate will 
be employed to stimulate the production of growth factor(s), most 
preferably about 0.03-0.2 mmole/l 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 Ser. No. 07/418,549 and in Borch et al. (U.S. Pat. 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 (e.g., mice) 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. Thus, 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 therapeutic effect of the dithiocarbamic compounds described above is 
not limited to treating damage caused by platinum-containing drugs. 
Mammals given platinum-free myleosuppressive antineoplastic drugs, 
subjected to irradiation, or that suffer from blood disorders such as 
aplastic anemia, can also be successfully treated with an effective amount 
of one of these dithiocarbamate salts or dimers (or, less preferably, 
acids, i.e. where M=H). 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. 
As will be explained subsequently, guidelines for converting these dosage 
units into mg/m.sup.2 have been discovered, both for large mammals, such 
as humans, and small mammals, such as mice. In principle, mg/m.sup.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. 
These very low dosages are believed to be well below stoichiometric levels 
and bear more resemblance to amounts at which catalysts are employed. 
Surprisingly, improvement in the blood-forming function of normal bone 
marrow is rather minimal when the dithiocarbamic compounds of this 
invention are administered to a healthy mammal. However, very significant 
improvements in bone marrow function are observed when the bone marrow of 
the mammal has been damaged, e.g., by administration of antineoplastic, 
e.g., cytotoxic drugs, by irradiation or by certain diseases. Accordingly, 
the therapeutic method of this invention applies primarily to mammals who 
have already suffered some myelosuppression effects. However, because 
there may be some time delay involved in observing the beneficial effects 
of this invention, it is possible to administer the dithiocarbamic 
compound more or less simultaneously with the myelosuppression-causing 
agent (i.e., the drug or radiation or the like). Typically, 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.sub.1 R.sub.2 NCSSM, wherein M is a 
pharmaceutically acceptable cation, and R.sub.1 and R.sub.2 are lower 
aliphatic or hydroxy-substituted lower aliphatic groups (e.g., a 
polyhydroxysubstituted lower C.sub.3 -C.sub.6 -alkyl group or a (C.sub.2 
-C.sub.6)-alkyl). The preferred route of administration of these compounds 
(particularly when M is a metallic cation) 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. In the case of the dimers (e.g., disulfiram), which are 
far less water soluble, the preferred route of administration is oral.

DETAILED DESCRIPTION 
Most of the discussion which follows is related to the use of 
dithiocarbamates to produce bone marrow cell growth factors or to protect 
against the bone marrow toxicity of anti-cancer drugs or radiation. 
However, it will be understood that 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. For example, 
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. As noted previously, 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. Although 
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 granulocyte/macrophage 
progenitor cell (GM-CFC) assay. 
Mechanistic studies done in connection with this invention reveal that 
anticancer drugs which inhibit tumor growth through interference with DNA 
synthesis (and which have the unfortunate effect of interferring with DNA 
synthesis in bone marrow also) are significantly modulated in their effect 
upon DNA synthesis in bone marrow when the dithiocarbamate is administered 
after the anti-cancer drug, e.g., three hours afterward. The mechanism of 
bone marrow protection provided by the dithiocarbamates is different from 
that involved in the reversal of other toxicities (e.g., kidney toxicity) 
and is not dependent upon stoichiometric displacement of platinum from 
biochemical structures. For example, the basis for the present process 
claims is our discovery that dithiocarbamates selectively stimulate 
proliferation of cultured bone marrow cells in vitro. However, 
dithiocarbamates such as sodium diethyldithiocarbamate (DDTC) do not alter 
the number of bone marrow cells proliferating in vivo in the absence of 
myelotoxic insult. 
TREATABLE BONE MARROW DAMAGE 
As noted previously, 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 busulfan, or heavy metal complexes 
(such as the platinum complexes discussed previously), and compounds 
containing the 2-chloroethyl group (typically, a 2-chloroethyl group 
attached to a nitrogen atom). There are compounds presently in clinical 
use which fall into none, or into more than one of these categories. For 
example, an antibiotic derivative of a 2-chloroethyl-containing compound 
or a cytotoxic agent can be a DNA synthesis inhibitor and/or an alkylating 
agent. In some cases, the mode of action of an antineoplastic drug is 
unknown, e.g., in the case of dacarbazine. 
"Adriamycin" (Doxorubicin hydrochloride) is an example of 
Streptomyces-produced antibiotic derivative which is known to cause bone 
marrow suppression effects, primarily of leukocytes. Hence, careful 
hematologic monitoring is required when this drug is being administered to 
produce regression in neoplastic conditions. 
There is a considerable variety of antineoplastic agents which have the 
2-chloroethyl (i.e., the betachloroethyl) group, typically attached to a 
nitrogen atom. Among these are lomustine. Some of these compounds are 
derivatives of L-amino acids, some are derivatives of steroids, some are 
monocyclic compounds, some are aliphatic amine derivatives, and still 
others are urea derivatives (including nitrosourea derivatives). Compounds 
of the nitrosourea type typically have the following formula: 
EQU C1--CH.sub.2 CH.sub.2 --N(NO)--CO--NH--R* 
wherein 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 1-3-bis(2-chloroethyl)-1-nitrosourea, also known as BCNU or BiCNU 
or carmustine. 
In antineoplastic drugs containing the 2-chloroethyl group, the 
bis-(2-chloroethyl)-amino functional group is particularly common, e.g., 
as in chlorambucil or cyclophosphamide. This bis-substituted group has the 
formula (ClCH.sub.2 CH.sub.2).sub.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. 
Of the agents which inhibit DNA synthesis or crosslink DNA molecules, the 
platinum (II) and (IV) compounds are among the most promising for clinical 
use. For a discussion of the types of platinum-containing drugs 
contemplated by Borch for use in combination with dithiocarbamic 
compounds, see (in addition to the three Borch patents) U.S. Pat. No. 
4,053,587 (Davidson et al.), issued Oct. 11, 1977; U.S. Pat. No. 4,137,248 
(Gale et al.), issued Jan. 30, 1979; U.S. Pat. No. 4,562,275 (Speer et 
al.), issued Dec. 31, 1985; U.S. Pat. No. 4,680,308 (Schwartz et al.), 
issued Jul. 14, 1987, and similar references appearing in both the patent 
and scientific literature, e.g., the series of papers regarding platinum 
treatment of tumors and resulting side effects in Cancer Treatment 
Reports, 63, 1433 (1979). The compound "cisplatin" (cis-dichlorodiammine 
platinum [II]) is very effective against testicular and ovarian tumors but 
has been found to have myelosuppressive effects in 25-30% of patients 
treated with this drug. More recent developments in platinum (II) and 
platinum (IV) anticancer drugs have produced compounds which are not only 
very effective against tumors but are also substantially free of side 
effects other than myelosuppression. Cisplatin, on the other hand, has 
significant kidney toxicity effects as well as bone marrow toxicity. 
Of 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.sub.3 - and C.sub.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", 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 antitumor utility, e.g., "TNO-6" 
and "CBDCA" (see U.S. Pat. No. 4,137,248) also showed increased bone 
marrow toxicity. These otherwise desirable Pt(II) compounds can be 
characterized by the formula: 
EQU (R'NH.sub.2)(R"NH.sub.2)Pt(X.sup.1)(X.sup.2) 
where X.sup.1 and X.sup.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.sub.1 and X.sup.2 preferably are SO.sub.3 H or 
--CO.sub.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 
The term "dithiocarbamic compounds" or dithiocarbamates as used in this 
application is intended to refer to compounds containing the functional 
group R.sub.1 R.sub.2 N--CS--S--, wherein R.sub.1 and R.sub.1 are the same 
or different and represent different aliphatic or cycloaliphatic or 
heterocycloaliphatic groups, e.g., (C.sub.1 -C.sub.6)alkyl, (C.sub.5 
-C.sub.10)cycloalkyl or five-to ten-membered heterocyclic groups, 
unsubstituted or substituted by hydroxyl. One of the two groups, R.sub.1 
and R.sub.2, but not both, can be hydrogen. Alternatively, R.sub.1 and 
R.sub.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. 
When the group R.sub.1 R.sub.2 N--CS--S-- is part of a dimer such as 
disulfiram, the dangling valence bond is linked to a group of the formula 
--S--CS--NR.sub.3 R.sub.4, wherein R.sub.3 and R.sub.4 are defined in the 
same manner as R.sub.1 and R.sub.2. When the group R.sub.1 R.sub.2 
N--CS--S-- is an anion, 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.sup.+, K.sup.+, Zn.sup.++ and the like. In the 
case of the dithiocarbamic acids, the group R.sub.1 R.sub.2 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, N.Y., 1962. As explained in Chapter 2 of Thorn et 
al., the preparation of dithiocarbamates is very simple. The compounds of 
the formula R.sub.1 R.sub.2 NCSSH or R.sub.1 R.sub.2 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. Thus, for example, sodium dimethyl dithiocarbamate is formed 
from CS.sub.2, NaOH and dimethylamine. See Thorn et al., page 14, and the 
references cited therein. Other typical dithiocarbamic compounds disclosed 
and characterized in Thorn et al. 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, cyclohexylamyldithiocarbamates, 
dibenzyl-dithiocarbamates, sodium dimethylene-dithiocarbamate, various 
pentamethylene dithiocarbamate salts, sodium pyrrolidine-N-carbodithioate, 
sodium piperidine-N-carbodithioate, sodium morpholine-N-carbo-dithioate, 
alpha-furfuryl dithiocarbamates and imidazoline dithiocarbamates. 
Another interesting type of dithiocarbamate which appears to have 
significant biovailability and biocompatibility includes compounds wherein 
R.sub.1 of the structure R.sub.1 R.sub.2 N--CS--S-- is a 
hydroxy-substituted or, preferably, a polyhydroxy-substituted lower alkyl 
group having up to 6 carbon atoms. For example, R.sub.1 can be 
HO--CH.sub.2 --CHOH--CHOH--CHOH--CHOH--CH.sub.2 --. In such compounds, 
R.sub.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.sup.2 is H, methyl, or ethyl. Accordingly, 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. 
The term "lower" (as in "lower alkyl" or "lower aliphatic"), as used in 
this discussion, 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 diethyldithiocarbamate (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. Patients complain of flushing and 
tightness in the chest during infusion of NaDDTC, and they develop 
symptoms of acute anxiety. These symptoms subside rapidly and without 
sequelae after the infusion is stopped, and the symptoms can be alleviated 
somewhat (but not abolished) by pretreatment sedatives. In the scientific 
literature, there are occasional references to analogous effects in rats, 
and these effects are sometimes referred to as the "rat rage" syndrome. A 
major advantage of this invention is that the "rat rage" syndrome can be 
avoided entirely due to the surprising efficacy of dosage units of this 
invention. 
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. 
Other preferred 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.sub.1 and/or 
R.sub.2 of the formula R.sub.1 R.sub.2 N--CS--S-- is a beta-hydroxyethyl. 
Generally speaking, the greater the solubility in polar solvents 
(particularly in aqueous media), the more convenient the administration of 
the dithiocarbamic myelosuppression treatment agent can be, because 
parenteral administration is particularly preferred in the method of this 
invention, and solutions (particularly aqueous solutions) are more 
convenient to administer than suspensions. 
For this reason, the monomeric dithiocarbamic compounds are preferred over 
the dimeric analogs. 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. 
Other 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. However, the dosage units of this 
invention are most effective by the intravenous route. 
DOSAGE UNITS AND FORMS 
It is very common in pharmacology to express dosage units in mg/kg (i.e., 
mg/kg of body weight) or, if a continuing series of doses over many days 
is contemplated, mg/kg per day. A mg/kg dosage unit is reasonably constant 
for any given species of mammal. However, 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. In 
principle, then, a dosage expressed in mg/m.sup.2 would be roughly 
equivalent in all species, regardless of body area, i.e., an ED.sub.50 of 
100 mg/m.sup.2 in a human would also be 100 mg/m.sup.2 in a mouse. To 
convert mg/kg to mg/m.sup.2, one multiplies by a constant for the desired 
species which is a function of the surface area of a member of that 
species, thus: 
EQU Dose in mg/m.sup.2 =Constant.times.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 the mouse constant. 
The dosage unit for NaDDTC administered to mice to ameliorate the kidney 
toxicity of Cisplatin (750 mg/kg, preferably &gt;200 mg/kg) works out to be, 
for example, 3.0.times.200 mg/kg=600 mg/m.sup.2, more typically 
3.0.times.300 mg/kg=900 mg/m.sup.2. Theoretically, then, the typical human 
dosage unit would be 900 mg/m.sup.2 divided by 37=about 25 mg/kg. In other 
words, theory would predict that the human dose in mg/kg would be about 
one-twelfth of the dose for mice. In actual practice, however, it has been 
found that the human dose of 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. In the present invention, a dosage of 0.3 
mg/kg (1 mg/m.sup.2) can 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.sup.2, 
more preferably 130 mg/m.sup.2, regardless of species. For all species, 
the dosage of 130 mg/m.sup.2 is ample and may be unnecessarily large. 
Suitable dosage units can be less than 90 mg/m.sup.2 or, if desired, less 
than 75 mg/m.sup.2. For humans, dosaqe 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 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. 
In the treatment of myelosuppression, dithiocarbamic treatment agents of 
this invention 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. To obtain a typical logarithmic 
dose-response curve, 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.sub.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 .mu.g/kg, e.g., about 1 
.mu.g/kg), and significant protection appears to be obtained, even in 
mice, at dosages as low as 3 .mu.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. When the dosage 
units are in mg/m.sup.2, a useful range is, for example, 1-200 mg/m.sup.2, 
more preferably about 1-75 mg/m.sup.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. 
Alternatively, 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.) 
Alternatively, 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. 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. Other suitable 
pharmaceutically acceptable carriers are available to those skilled in the 
art. 
The principle and practice of this invention is illustrated in the 
following non-limiting Examples. 
EXAMPLE I 
BDF.sub.1 mice were used and the drugs were administered 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 evaluated using the spleen colony (CFU-S) assay; toxicity to 
granulocyte progenitors was evaluated using an in vitro clonogenic 
(CFU-GM) assay. To provide controlled studies, mice were randomly divided 
into four groups of four animals each; one group served as a no-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. 
For the CFU-S assay, 5-15.times.10.sup.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.sup.5 
cells injected and are reported as the percent of colonies formed compared 
to the control group. 
For the CFU-GM assay, 2-4.times.10.sup.4 bone marrow cells from the treated 
groups were plated on soft agar. After incubating for 7 days, the colonies 
containing at least 50 cells were counted; in representative experiments, 
the colonies were removed and the cell type determined. The data 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. In the drug-only group, dose-dependent 
toxicity toward both CFU-S and CFU-GM was observed for carmustine (BCNU) 
and adriamycin. In the drug and DDTC group, DDTC provided significant 
protection against BCNU toxicity to both stem cells and granulocyte 
progenitors at all doses of BCNU tested. In the case of adriamycin, 
reduction of toxicity was observed at all doses but was less impressive at 
the highest adriamycin dose tested. 
The situation in the case of mitomycin (an anticancer drug of the 
antibiotic type) is more complicated because it is particularly difficult 
to prevent or reverse the myelosuppressive effects of this drug. 
Very good results were obtained when the drug+DDTC group was given 
carboplatin (a platinum-containing anticancer drug) followed by various 
doses of DDTC. Carboplatin given to the drug-only group resulted in mice 
having CFU-S values which were only 10% of the control group level. When 
the CFU-S assay shows 30% or more of the value of the control (no 
treatment) group, this is considered indicative of very good activity 
against myelosuppression. The 30% level in the drug+DDTC group was 
achieved with an iv dose of 30 mg/kg of NaDDTC, but 40% of the control 
CFU-S level was also achieved with an iv dose of only 0.3 mg/kg of NaDDTC. 
In the experiments summarized in Table I (which were conducted according to 
the procedure described above), the dose of NaDDTC was 300 mg/kg of body 
weight, which appears to be excessive, but which illustrates the efficacy 
of dithiocarbamate, vis-a-vis damage from platinum-free drugs. Both in 
Part A (drug=BCNU) and in Part B (drug=adriamycin), data are given for the 
"DDTC group", the "drug-only group", and the "drug and DDTC group". These 
data are set forth in Table I, below. 
TABLE I 
______________________________________ 
EFFECT OF NaDDTC ON DRUG-INDUCED 
MYELOSUPPRESSION 
Drug Dose Mouse 
(mg/kg) Group CFU-S (%) CFU-GM (%) 
______________________________________ 
Part A 
Drug: BCNU 
-- DDTC 102 .+-. 2 
102 .+-. 1 
20 Drug-only 47 .+-. 6 83 .+-. 2 
20 Drug and 57 .+-. 12 
99 .+-. 2 
DDTC 
-- DDTC 101 103 .+-. 2 
40 Drug-only 30 .+-. 1 43 .+-. 2 
40 Drug and 50 .+-. 1 83 .+-. 2 
DDTC 
-- DDTC 114 102 .+-. 2 
65 Drug-only 19 .+-. 2 25 .+-. 1 
65 Drug and 49 .+-. 11 
64 .+-. 1 
DDTC 
Part B 
Drug: Adriamycin 
-- DDTC 106 102 
18 Drug-only (21) 37 .+-. 6 
18 Drug and (42) 45 .+-. 2 
DDTC 
-- DDTC -- 102 .+-. 1 
24 Drug-only 40 32 .+-. 1 
24 Drug and 52 42 .+-. 2 
DDTC 
-- DDTC -- 102 
32 Drug-only 29 .+-. 8 20 .+-. 5 
32 Drug and 57 .+-. 7 28 .+-. 2 
DDTC 
______________________________________ 
EXAMPLE II 
The following experiments were conducted to demonstrate the production of 
bone marrow cell growth factor(s) with DDTC. 
A. Materials and Methods 
Cis-diammine(cyclobutanedicarboxylato)platinum (II), or "CBDCA" was 
obtained from Johnson-Matthey, Inc. (Malvern, Pa.). Sodium 
diethyldithiocarbamate (DDTC) was obtained from Sigma Chemical Company 
(St. Louis, Mo.). Fischer's medium, minimum essential medium, alpha 
modification (.alpha.-MEM), L-glutamine, pokeweed mitogen, sodium 
bicarbonate (7.5% solution), gentamicin, penicillin/streptomycin solution, 
and antibiotic/antimycotic solution were purchased from GIBCO (Grand 
Island, N.Y.). Horse serum and fetal bovine serum were purchased from 
Hyclone Laboratories (Logan, Utah). Salmonella typhosa lipopolysaccharide 
B (LPS) was purchased from Difco Laboratories (Detroit, Mich.). 
Methylcellulose (4A premium grade) was provided by Dow Chemical Company 
(Midland, Mich.). Falcon Petri plates and microscope slides were obtained 
from Fisher Scientific Company (Springfield, N.J.). All other plastic 
culture supplies and test tubes were obtained from VWR (Rochester, N.Y.). 
B. Experimental Animals 
Male 6- to 8-week-old C57BL/6J.times.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. 
C. Establishment of Murine Long-term Bone Marrow Cultures (LTBMC) 
Long-term bone marrow cultures were established according to J. S. 
Greenberger, in Hematopoiesis, D. W. Golde, ed., Churchill Livingstone, 
Edinburgh (1984) at pages 203-242. One ml of growth medium [Fisher's 
medium (pH 7.0) supplemented with 25% horse serum, 100 U/ml penicillin G, 
and 100 .mu.g/ml streptomycin] was used to aseptically flush marrow cells 
from one murine tibia and femur into a flask containing 9 ml of growth 
medium. The cultures were maintained in a fully humidified incubator, 5% 
CO.sub.2 atmosphere, at 33.degree. 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.sup.-5 M 
hydrocortisone sodium hemisuccinate, to facilitate development and 
maintenance of the adherent cells. 
Cultures of the stromal bone marrow cells were established in the same 
fashion. However, the supplemental horse serum (25%) was replaced with 20% 
fetal bovine serum as a supplement to Fischer's medium (pH 7.0) and the 
antibiotic solution as described above. These culture conditions do not 
allow survival of CFU-GEMM or GM-CFC (confirmed by removing the adherent 
cells from three cultures and testing for the presence of colony forming 
units-granulocyte/erythrocyte/macrophage/megakaryocyt(CFU-GEMM) or 
GM-CFC). In all other respects, the cultures were initiated and maintained 
as described above. 
D. Granulocyte/Macrophage Progenitor Cell (GM-CFC) Assay 
This assay was carried out using the method of T. K. Schmalbach et al., 
Cancer Res., 49, 2574 (1989), but with the following modifications. Bone 
marrow cells were harvested from untreated mice, and in the LTBMC 
experiments, the PWM-SCCM was replaced with 500 .mu.l of supernatant 
harvested from the drug treated (or control) LTBMC. Granulocyte/macrophage 
colonies&gt;50 cells were counted on day 7 with the aid of a dissecting 
microscope. 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 (.alpha.-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 pokeweed 
nitrogen-stimulated spleen cell-conditioned medium (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. 
E. Determination of Colony Stimulating Activity (CSA) in Media of 
Drug-Treated LTBMC 
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 .mu.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 .mu.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.times.g for 5 min.). The supernatants were 
subsequently evaluated for colony-stimulating activity in the GM-CFC assay 
as described above. 
A similar procedure was used to determine the response of drug-treated 
cultures exposed to mitogen stimulation. In these experiments, Salmonella 
typhosa lipopolysaccharide B (5 .mu.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. 
F. Results 
After 5 weeks, the LTBMC were treated with DDTC with or without prior 
treatment with CBDCA. At various times, the supernatants were removed and 
the colony-stimulating activity (CSA) of each supernatant was assessed by 
using it to replace the pokeweed mitogen-stimulated spleen cell 
conditioned medium in the GM-CFC assay. Basal levels of 
granulocyte/macrophage colony-stimulating activity (CSA) in control 
supernatants varied with each experiment, since differences in serum 
constituents are known to affect the ability of LTBMC to support 
hematopoiesis. Enhancement of CSA in three separate experiments is 
summarized in Table II. 
TABLE II 
______________________________________ 
Enhancement of GM-CSA by Supernatants Removed from 
LTBMC Treatment Aqent* 
Pokeweed 
Removal CBDCA + Mitogen 
Time (hr) 
CBDCA DDTC DDTC (PWM)** 
______________________________________ 
24 1.0 .+-. 0.1 
3.9 .+-. 0.7 
2.6 .+-. 0.4 
8.6 .+-. 1.9 
48 1.2 .+-. 0.2 
3.4 .+-. 0.7 
3.0 .+-. 0.6 
6.6 .+-. 2.2 
72 1.1 .+-. 0.1 
3.3 .+-. 0.5 
2.9 .+-. 0.5 
8.2 .+-. 2.2 
96 0.8 .+-. 0.1 
4.7 .+-. 1.7 
3.9 .+-. 1.1 
8.9 .+-. 3.1 
Combined 1.0 .+-. 0.1 
3.8 .+-. 0.5 
3.1 .+-. 0.4 
8.1 .+-. 1.2 
______________________________________ 
*Results are ratio of colonies/10.sup.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. 
**Positive control. 
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 CSA 
either alone or when added just prior to DDTC treatment. DDTC enhanced CSA 
at concentrations from 100-1000 .mu.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., 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. Addition of hydrocortisone 
hemisuccinate to the DDTC-treated cultures neither enhanced or diminished 
DDTC-induced CSA compared to treatment with DDTC alone (data not shown). 
Supernatants from cultures treated with a maximally stimulating 
concentration of LPS (5 .mu.g/ml) induced formation of 195 
colonies/10.sup.5 cells. Neither DDTC, CBDCA, nor the combination of CBDCA 
and DDTC significantly changed the CSA of these supernatants (190-210 
colonies/10.sup.5 cells). These results demonstrate that DDTC is inducing 
production of colony-stimulating factor(s) that is not additive with 
respect to stimulation by either hydrocortisone or LPS. 
EXAMPLE IV 
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. Thus, LTBMC containing stromal cells 
(including monocytes/macrophages) 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. Untreated supernatants from these cultures 
had greater CSA compared to those obtained from the complete LTBMC, and 
DDTC treatment enhanced CSA approximately twofold compared to untreated 
cultures. Again, CBDCA treatment had no significant effect on untreated or 
DDTC treated cultures. These data indicate that DDTC stimulation of CSA is 
most pronounced during the first 24 hours after treatment. This was 
confirmed by comparing the CSA of supernatants collected over varying time 
intervals. CSA was significantly enhanced by DDTC in supernatants collected 
between 0-8 hours and 8-24 hours after DDTC treatment but was not 
significantly different from untreated supernatants obtained during later 
time intervals (data not shown). 
The results of Examples II-IV indicate that DDTC modulates hematologic 
toxicity by inducing stromal cell production of a factor or factors that 
stimulate hematopoiesis. Although 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. These results are consistent with a mechanism in which 
DDTC augments rather than initiates a proliferative response. 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. Although the identity of the factor(s) responsible for the CSA 
induced by DDTC is not known, several cytokines may be potential 
candidates. For example, GM-CSF and granulocyte colony stimulating factor 
G-CSF may play a role in the DDTC response, but their effects may be 
secondary to release of another cytokine such as IL-1.alpha., IL-1.beta., 
IL-6, IL-3, or mixtures thereof. The concentration of tumor necrosis 
factor has also been found to be increased in culture. 
Thus, the production of one or more growth factors (which factor or factors 
have G/M cell CSA) 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 compound of the 
formula I (R.sup.1 R.sup.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. 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 concentrate and purify cytokines. 
Accordingly, 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 R.sup.1 
R.sup.2 N(CS)SM. Hence, 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. 
For example, the stimulation 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.sup.1 R.sup.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. That is, 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. 
All of the documents cited hereinabove are incorporated by reference 
herein. The invention has been described with reference to various 
specific and preferred embodiments and techniques. However, it should be 
understood that many variations and modifications may be made while 
remaining within the spirit and scope of the invention.