Thiol activation of cytotoxic and auxin-herbicidal agents and root formation stimulation

INTRODUCTION
TECHNICAL FIELD
The field of this invention is the killing of proliferating cells and enhancing root formation.
BACKGROUND
There are many situations where one wishes to modulate cellular growth. Since all of life is dependent upon cellular regeneration, there is substantial interest in being able to modulate cellular growth in the plant and animal kingdoms. In the case of plants, one wishes to encourage the growth of desirable plants, such as plant crops and flowers, while at the same time one wishes to discourage the growth of weeds, both pre-germination and post-germination of the desired plant. In many situations one wishes to kill all vegetation, such as highway borders, pathways, graveled areas, and the like. However, many of the herbicides which are used today pollute the environment in introducing undesirable elements into the soil, which can then be leached into aquifers, streams and rivers. There is, therefore, substantial interest in being able to reduce the amount of herbicidal use without further pollution of the environment.
There is also the need to root stock to expand desired plants, particularly woody stock. Better methods are needed to increase the efficiency and reduce the time for rooting.
Finally, there is the proliferation of cells in the animal kingdom. The development of the various species from a single fertilized cell to the mature adult requires rapid proliferation. However, during the life of the organism, there are many situations where rapid proliferation is undesirable. Various diseases associated with rapid proliferation include cancers, psoriasis, restenosis, and the like. Many of the agents used for treating these diseases, are cytotoxic agents which attack all proliferating cells and frequently have substantial side effects in addition to their cytotoxic activity.
Various techniques have been employed to reduce the systemic influences of these cytotoxic drugs. Immunotoxins employ a toxin joined to an antibody, where the antibody is specific to varying degrees for a marker present on cancer cells. Another technique which is limited to physical access to the tumor employs the cytotoxic drug in a matrix which reduces the translocation of the cytotoxic drug from the site of the lesion. Other techniques have involved use of liposomes with agents, such as antibodies, to direct the liposomes to the target, where the lumen of the liposome comprises a cytotoxic agent. However, none of these techniques has found universal acceptance. So long as one continues to use the cytotoxic drugs in systemic fashion, there is substantial interest in finding ways to diminish the amount required for an effective dosage as a cytotoxic agent.
RELEVANT LITERATURE
The classic method of root induction is to utilize an auxin shock. Lane (1978) Plant Sci. Lett. 13:281-285 and Moncousin (1992) Rooting of in vitro Cuttings. In: Biotechnology in Agriculture and Forestry (YPS Bajag, ed.) Springer Verlag, Heidelberg, 17:231-261. Standardi and Romani (1990) Hort. Sci. 25:1435-1436 teaches auxin shock for rooting and employs cysteine.
SUMMARY OF THE INVENTION
Thiol compounds are employed as adjunctive agents to enhance modulation of cellular proliferation. Particularly, the thiol agents are found to enhance cytotoxicity, both in plants and animals, as well as enhancing root formation in conjunction with auxins or auxin shock.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for modulation of cellular proliferation in plants and animals. Particularly, the thiol compounds are used in conjunction with cytotoxic agents to reduce the amount of the cytotoxic agent required for cytotoxicity. By combining the thiol compound with the cytotoxic agent, either locally or systemically, substantially smaller amounts of cytotoxic agent are required in order to achieve desired levels of cytotoxicity. In mammals, one may use combinations of cytotoxic agents plus thiol compound to control the growth of cells in proliferative diseases, such as cancer. The combination may be administered systemically or locally.
CYTOTOXICITY OF PLANT CELLS
The control of undesired vegetation will be considered first. The subject compositions comprise an auxin-type herbicide, e.g. 2,4-dichlorophenoxyacetic acid and triclopyr, optionally in conjunction with ammonium nitrate, and the thiol compound. The auxin type herbicide may be used as an acid derivative, particularly salts and esters, where the ester group may be of from 1 to 12 carbon atoms and the salts may include amino salts of from 0 to 12 carbon atoms. These derivatives are well known in the literature and commercially available.
A wide variety of thiol compounds may be employed, the primary consideration will be economics, convenience, effect on the environment, ease of formulation, handling characteristics, physiological activity toward mammals, and the like. A number of thiol compounds are available and may be used, such as cysteine, glutathione, dithiothreitol, thiolpropionic acid, etc. For the most part, the compounds will generally be from about 2 to 12, usually 2 to 10 carbon atoms, and may have other functionalities, such as oxy, oxo, including esters and amides, amino, etc. As indicated above, the choice of mercaptan is not critical to this invention, so long as it is not too volatile, does not have an offensive odor, and can be readily handled.
Depending upon the particular auxin-type herbicide employed, one will normally use less than about 60%, usually less than about 50%, and generally more than about 20% of the dosage necessary for herbicidal activity of the auxin-type herbicide, by itself, or in conjunction with ammonium nitrate. The amount employed will vary depending upon the particular vegetation involved, the degree to which one wishes to kill the vegetation and the rapidity with which the vegetation is to be killed. Thus, where it is sufficient that only a portion of the wild vegetation be killed, milder formulations may be employed, where rapid complete death is desired, the formulations will be stronger.
Generally, the amount of herbicide will vary with the particular herbicide, generally ranging from about 1 to 6 pounds per acre, preferably from about 1 to 5 pounds per acre, more preferably from about 2 to 4 pounds per acre. When ammonium nitrate is present, it will generally range from about 0.025 to 2 pounds per acre, more usually from about 0.05 to 1.5 pound per acre. The thiol compound will vary to some degree based on the particular thiol compound but will generally be in the range of about 0.01 to 2 pounds per acre, more usually in the range of about 0.025 to 1.5 pounds per acre, and preferably 0.05 to 1 pound per acre. Formulations of particular interest for total vegetative kill include 3 to 5 pounds per acre of the herbicide, particularly trlclopyr, 1 to 2 pounds per acre of ammonium nitrate, and 0.05 to 0.25 pounds per acre of cysteine.
The subject formulations can be formulated in a variety of ways, such as dispersible dry powders, pellets, solutions, or the like. In the formulation, the weight ratio of thiol compound to herbicide will be generally in the range of about 1:2-100, more usually in the range of about 1:10-100, while the ratio of the ammonium nitrate to herbicide will generally be in the range of about 1:1.5-100 more usually 1:2-50.
Other additives and excipients may be included in the formulation, depending upon the manner in which the formulation will be applied. Other additives which may be included in minor amounts include spreading agents, penetrating agents, surfactants, emulsifiers, etc., with the other components in appropriate relationship.
The subject compositions can be used pre-emergent, and may find application post-emergent, particularly as a spray, although pellets may also be used and scattered. For the spray, the herbicide will generally be about 1 to 10, more usually 2 to 8, preferably 3 to 6 pounds per 100 gallons, the ammonium nitrate if present, will generally be 0.5-5, usually about 2 to 4, pounds for 100 gallons and the thiol compound will generally be about 0.1 to 1, more usually about 0.1 to 0.5 pounds for 100 gallons. The composition may be provided as a concentrate, for further dilution, where the herbicide will be from about 1 to 5 pounds per gallon, usually employing an emulsifier.
In some instances, combinations of herbicides may be employed, such as combinations of triclopyr, picloram, 2,4-D, glyphosate, clopyralid, fanoxaprop-ethyl, oxadiazon, bromoxynil, propanil, dicamba, and the like, where the additional herbicide may provide specific advantages for particular applications. Generally, the weight ratio of the secondary herbicide(s) to the primary auxin type herbicide described above, will generally be from about 0.01-0.5:1, more usually from about 0.05-0.25:1.
When spraying, the application rate will generally provide the amount of herbicidal composition indicated above. Conveniently, one may provide for formulations which allow for spraying at about 10 to 50 gallons per acre, more usually at about 15 to 35 gallons per acre.
The subject formulations are particularly applicable to woody trees and brush, where the species are resistant to the auxin-type herbicide, by itself or in conjunction with other herbicides. The subject compositions are found to be effective for non-evergreen roadside vegetation and deciduous wood species, such as ash and hard maple.
Species that are controllable and very sensitive to the herbicides include briars (blackberry, raspberry, etc.), mulberry, walnut, sumac, sassafras, box elder, buckeye, cottonwood, wild grape and poison ivy; species that are sensitive include red bud, elm, honey locust, black locust, hackberry, willow, and white oak; species of intermediate sensitivity include hard maple and black cherry; and species that are resistant include juniper and ash, all of which can be controlled by the subject formulation.
CYTOTOXICITY OF MAMMALIAN CELLS
The thiol compounds act via alterations in the activity patterns of a growth-related NADH oxidase (disulfide-thiol interchange protein). This activity is altered in cancer and a protein earing this altered activity circulates in the blood. The circulating form of the cancer associated altered form of the NADH oxidase has been designated as t-NOX and is useful as a diagnostic procedure for cancer. The thiol compounds find use as an enhancer of in conjunction with diagnostic uses of drugs acting as inhibitors of t-NOX. With patient sera treated with 1 .mu.M to 100 .mu.M capsaicin, the maximum inhibition of NADH oxidation with serum is 15 to 40%. However, if the capsaicin treatment is followed by 0.1 to 10 .mu.M reduced glutathione, a pronounced additional transient inhibition of 5 to 10 min duration is observed. This inhibition is not observed with sera of normal patients.
The thiol compounds can also find use for reactivation of sample preparations that have been frozen prior to use in a diagnostic assay for cancer associated t-NOX. Addition of 0.1 to 10 .mu.M of oxidized glutathione to such preparations restores the enzymatic activity. Inclusion of up to an equivalent amount of reduced glutathione with the serum samples during storage will also help maintain enzyme activity. Comparable amounts of oxidized thiol compounds other than oxidized glutathione also find use.
For use in mammals to inhibit cellular hyperproliferation particularly in association with cancer, various cytotoxic agents may be used in conjunction with thiol compounds. The subject compositions may be used with any type of cancer, whether solid or dispersed, including carcinomas, sarcomas, lymphomas, melanomas, and the like. The cancers may be associated with various tissues and organs, such as blood, breast, prostate, ovary, lung, kidney, liver, eye, skin, pancreas, bone, bone marrow, leukocytes, etc.
Chemotherapeutic drugs which may be employed include enzyme inhibitors, proliferation inhibitors, lytic agents, DNA synthesis inhibitors, membrane permeability modifiers, DNA intercalators, antimetabolites, or the like. Illustrative drugs include chlorambucil, melphalan, busulfan, carmustine, lomustine, streptozotocin, thiotepa, dacarbazine, methotrexate, 5-flurouracil, cytarabine, azaribine, mercaptopurine, thioguanine, vinblastine, vincrinistine, actinomycin D, adriamycin, bleomycin, mithramycin, mitomycin C, L-asparaginase, cisplatin, sulfonylureas, capsaicin, and its derivatives, and the like. Of particular interest are drugs which inhibit a surface membrane NADH oxidase associated with the hyperproliferative state. These compounds include adriamycin, sulfonylureas, capsaicin, and the like.
The chemotherapeutic drug will normally be used in an amount of from about 10 to 80 weight percent, more usually from about 10 to 60 weight percent, particularly from about 20 to 60 weight percent of the normal chemotherapeutic dosage. The thiol compounds, depending upon the particular drug, may be formulated together with the chemotherapeutic drug or separately and combined immediately prior to administration or concomitantly or consecutively with administration of the chemotherapeutic drug. The amount of the thiol compound which finds use in proportion to the amount of chemotherapeutic drug will generally be optimized within the ratio of about 0.01 to 10, more usually from about 0.05 to 1 of thiol compound to chemotherapeutic drug. Thiol compounds of interest include cysteine, glutathione, thiolacetic acid, cysteine, 2-mercaptoethanol, dithiothreitol, mercaptamine, etc.. Since the thiol compounds will normally be physiologically acceptable and relatively large amounts of the thiol compound can be used without adverse physiological effect, once the minimum amount is defined for the desired therapeutic activity, one may use increasingly larger doses of the thiol compound without adverse effect and frequently without any further advantage.
The subject composition may be used both in vitro and in vivo. In many situations, it is desirable to inhibit growth of cancer cells in the presence of normal cells in culture. By augmenting the activity of the chemotherapeutic agent, with an agent such as the thiol compound which does not normally have adverse physiological effects, one can substantially reduce the adverse effect of the chemotherapeutic agent on the normal cells. In this manner, one can minimize the adverse effects on normal cells, while insuring the substantial absence or absence of tumorous cell growth. The chemotherapeutic agent and thiol additive may be maintained at a substantially constant concentration replenishing the culture medium on a regular basis, conveniently by substituting one half of the culture medium on a schedule of from 1 to 7 days.
For use in mammals, the subject compositions can be introduced intralesionally or parenterally, particularly intravascularly or intramuscularly. For intralesional administration, the subject compositions may be injected directly into the lesion, by themselves or in conjunction with vasoconstrictive agents, and/or matrices, such as collagen, fibrinogen, heparin, or the like, individually or in combination. See, particularly, U.S. Pat. No. 4,619,913. Generally, the amount of drug will range from about 0.01 to 30% of the composition depending upon the nature of the drug. Usually, the amount of cytotoxic drug administered to the tumor site will range from about 0.01 to 500, more usually about 0.05 to 300 mg/kg of the host. As illustrative, about 50 to 200 .mu.M of a chemotherapeutic sulfonylurea localized concentration is provided. The thiol concentration will therefore be in the range previously indicated in relation to the chemotherapeutic agent.
For intravascular administration, the formulation may be present in any convenient physiologically acceptable medium, to provide a systemic concentration of the chemotherapeutic agent in proportion to the normal dosage. Carriers may include water, saline, phosphate buffered saline, vegetable oils, ethanol, or the like. Concentrations may be varied widely, depending on the manner of administration, solubility of the chemotherapeutic agent, etc. Small amounts of other additives may be present, such as emulsifiers, stabilizers, buffers, and the like. In some instances, the subject formulation may be provided in the lumen of a liposome for slow or directed release.
ROOTING OF SHOOTS
Rooting is carried out in three phases. Phase 1 is induction (0 to 3 or 4 days.) Phase 2 is dedifferentiation and the formation of root meristems (1 to several weeks). phase 3 is growth and emergence of new roots (several weeks to 1 month). In some situations, especially with plantlests in vitro, the induction phase following the auxin shock is carried out in darkness (up to 5 days). Thereafter, rooting is either in continuous light or alternating light and dark cycles appropriate to the species. For stem cuttings, all 3 phases of rooting are carried out either in continuous light or under alternating dark and light cycles appropriate to the plant material.
In conjunction with rooting, thiols may be used as adjuvants to enhance root stimulation, particularly with difficult-to-root varieties. The thiol compounds described above, particularly cysteine and dithiothreitol, find use in this application in conjunction with auxins in the growth medium or auxin shock. Auxins include indole-3-acetic acid, indole-3-butyric acid, .alpha.-naphthaleneacetic acid, etc. Prior to auxin shock, the cuttings or shoots may be subject to maintenance in the dark from 0.5 to 2 weeks under mild conditions. The auxin shock may be carried out as appropriate, generally employing from about 0.5 to 5 mM of an appropriate auxin, conveniently 1-2 mMindole-3-acetic acid (IAA). The auxin shock is performed in a conventional manner, conveniently using an agarized solution and placing the microcutting into the solution for from 0.5 to 5, more usually from about 0.5 to 3, and preferably from 1 to 2 h. The timing may vary depending upon the particular species and its resistance to formation of roots. The microcuttings may be obtained from callus, tissue, intact plants or other appropriate plant parts or sources of growing tissues where formation of roots might be desirable. The auxin solution will be in a solvent comprising such other additives as minerals, vitamins, salts, buffers, detergents, stickers, wetting agents, spreaders and other growth enhancers.
Instead of auxin shock, a combination of auxin and thiol compound can be employed in the rooting medium. Various conventional rooting mediums may be employed such as Murashige and Skoog mineral solutions with conventional additives, such as inositol, thiamine, nicotinic acid, indolebutyric acid and a carbon source e.g. sucrose; a woody plant medium mineral solution (Lloyd and McCown,(1981) Int. Plant Prop. Soc. Proc. 30:421-427) supplemented with vitamins (Mullin et al. (1974) Phytopath. 64:1425-1429) and amino acids (Skoog (1944) Am. J. Bot. 31:19-24), with plant growth regulators, such as benzylaminopurine and alpha-naphthylacetic acids, with a carbon source and an amino acid source, such as Difco bactoagar. The amount of auxin in the rooting medium will be about 2 mM. The particular medium will be selected in accordance with conventional ways for the particular plant.
The thiol compound may be provided with the auxin shock or auxin continuously or intermittently during the first 10 days of maintenance in the shooting medium. After the auxin shock, the cuttings or shoots are rinsed and then introduced into an agar medium or distilled water comprising the appropriate composition. The thiol compound will generally be present in about 0.05 to 5 mM, preferably about 0.1 to 2 mM, where the response may vary with the particular thiol source and the time at which the thiol compound is provided. Usually, during growth, the thiol compound will be provided over at least one day and may be provided during the entire period, with some preference for the first one to three day on the six to nine day periods from the initiation or rooting stimulation with the auxin shock or auxin containing rooting medium. A wide variety of monocots and dicots both non-woody and woody plants, may be used with the subject formulation. Illustrative plants include apple, dipladenia, daphne, mung bean, soybean, Hedera, Forsythia, Salix, various Prunus species, Chrysanthemum, Poinsettia, Acacias, Acer, Alnus, Azalea, Bougainvillaea, Bouvardia, Castanea, Carica, Chaelomenes, Cercis, Citrus, Cornus, Corylus, Cotinus, Eucalyptus, Cydonia, Fagus, Gardenia, Garrya, Ginko b., Hamamelis, Hydrangea, Ilex, Kalmia, Malus, Pieris, Pyrus, Quercus, Ribes, Rosa, Skimmia, Syringa, Vaccinium, Viburnum, Vitis, Rubus, Spirae and various other fruit-bearing, ornamental and commercially-utilitarian woody species as well as flowers, vegetables and agricultural and horticultural crop species as well as conifers and evergreens.

The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
I. Effect of Thiol Compounds on Activity of Herbicides
A number of studies were performed to evaluate the effect of various combinations of auxin like compounds, with and without ammonium nitrate, and without a thiol additive and with different thiol additives on a variety of weeds and woody shrubs. These particular plant species are frequently found in areas where vegetation is undesired and there is a strong interest in maintaining the area substantially free of vegetation. The results of the studies are set forth in a series of tables, tables 1 to 9.
TABLE 1__________________________________________________________________________Comparison of triclopyr (Garlon-4) and the ammonium nitrate additivecombined with TR-III (Cysteine) on control of ash and hard maple.IN-126 test area. Treatments were applied August 10, 1993. Evaluationswere on October 14, 1993. Amount/40 gal waterAmmonium X-77 Defoliation, %Triclopyr nitrate surfactant TR-III Ash Hard Maple__________________________________________________________________________2 lb 1 lb 0.1 gal* None 60 .+-. 10 50 .+-. 102 lb 1 lb 0.1 gal 1 lb 90 .+-. 10 100 .+-. 0__________________________________________________________________________ *0.25% of the total spray mixture
TABLE 2__________________________________________________________________________Effect of cysteine in combination with 2,4-D and triclopyr on killof white clover (Trifolium repens). % Dead Applied Applied Applied Applied 5/31/94, 6/1/94, 6/6/94, 6/7/94, Cysteine Evaluated Evaluated Evaluated EvaluatedHerbicide 0.05 lb/A 6/6/94 6/22/94 6/19/94 7/5/94__________________________________________________________________________None - 0 0 0 0 + 0 0 0 02,4-D, - 30 20 15 205 lb/A + 50 40 30*** 30***Triclopyr, - 60 60 50 605 lb/A + 80* 80*** 70*** 100***__________________________________________________________________________ *90% kill on 6/12/94 and 100% kill on 6/19/94. **90% kill on 6/27/94. ***Contained 1 lb/A ammonium nitrate.
TABLE 3__________________________________________________________________________Comparison of cysteine and glutathione in combination with2,4-D and triclopyr on kill of white clover (Trifolium repens). % Dead Applied Applied 6/8/94, 6/9/94, Evaluated EvaluatedHerbicide Cysteine Glutathione Dithiothreitol 7/5/94 6/19/94__________________________________________________________________________2,4-D, -- -- -- 20 202 lb/A 1 lb/A -- -- 30 30 -- 1 lb/A -- 50 -- -- -- 1 lb/A -- 30Triclopyr, -- -- -- 90 402 lb/A 1lb/A -- -- 100 50 -- 1 lb/A -- 90 -- -- -- 1 lb/A -- 60__________________________________________________________________________
TABLE 4______________________________________Dose response of reduced glutathione, dithiothreitol andcysteine on herbicidal activity of 3.5 lb/A triclopyr onwhite clover (Trifolium repens). Applied 6/30**, 7/1/94***and 7/6/94****. Evaluated 7/20/94**, and 7/23/94****.% DeadRate/A Glutathione** Dithiothreitol*** Cysteine****______________________________________0 5 20 500.05 90 40 600.1 100 50 500.1* 100 50 300.5 80 40 601 80 50 40______________________________________ *+0.1 lb/A ammonium nitrate
TABLE 5______________________________________Enhancement of herbicidal activity of 5 lb/A triclopyr onwhite clover (Trifolium repens) by ammonium nitrate.Applied 6/16/94. Evaluated 6/26/94.Ammonium nitrate, lb/A % Dead______________________________________0 400.05 55______________________________________
TABLE 6______________________________________Dose response of cysteine in the presence of 2 lb/Atriclopyr and 1 lb/A ammonium nitrate on white clover(Trifolium repens).% DeadCysteine Applied 7/6/94 Applied 7/7/94Rate/A Evaluated 7/23/94 Evaluated 8/1/94 Average______________________________________0 80 50 70 670.025 100 80 90 900.05 80 60 90 770.1 90 60 80 770.5 100 60 80 801 100 80 80 87______________________________________
TABLE 7__________________________________________________________________________Control of common milkweed (Asclepias syriaca) and canada thistle(Cirsicum arvense) by triclopyr + ammonium nitrate + cysteine. % DeadRate/A, lb Milkweed Field Bindweed Ammonium (Asclepias Canada thistle (ConcolculusTriclopyr Nitrate Cysteine syriaca) (Cirsicum arvense) arvensis)__________________________________________________________________________2 -- -- 20 30 922 1 0.1 30 40 962 1 1 50 50 955 -- -- 80 70 955 0.05 0.05 100 50 98__________________________________________________________________________ 1 Applied 7/11/94. Evaluated 8/20/94. 2 Applied 7/12/94. Evaluated 7/25/94. 3 Applied 7/13/94. Evaluated 8/3/94.
TABLE 8__________________________________________________________________________Response to two rate of cysteine of white clover(Trifolium repens) in combination with ammonium nitrateand triclopyr. % Dead Applied Applied Appliedlb/A 7/20/94, 7/20/94, 7/25/94, Ammonium Evaluated Evaluated EvaluatedTriclopyr nitrate Cysteine 8/1/94 8/3/94 8/20/94 Average__________________________________________________________________________2 -- 0.05 45 45 35 402 1 1 50 45 40 452 1 -- 30 30 40 355 -- -- 50 50 35 45__________________________________________________________________________
TABLE 9______________________________________Response of three resistant weed species to two ratesof cysteine in combination with ammonium nitrate and triclopyr.lb/A % Dead Ammonium Canada CommonTriclopyr nitrate Cysteine thistle.sup.1 milkweed.sup.2 Chicory.sup.3______________________________________2 -- -- 40 45 602 1 0.05 50 60 802 1 1 40 20 801 -- -- 60 50 80______________________________________ .sup.1 Circisum arvense. Applied 7/26/94. Evaluated 8/20/94. .sup.2 Asclepias syriaca. Applied 7/27/94. Evaluated 8/20/94. .sup.3 Chicorium intybus. Applied 7/28/94. Evaluated 9/12/94.
TABLE 10__________________________________________________________________________Weed control by 2 lb/A triclopyr with or without ammonium nitrate orcysteine on wild carrot (Daucus carota),Canada thistle (Cirsicum arvense), whorled milkweed (Asclepiasverticillata), common milkweed(Asclepias syriaca), field bindweed (Convolvulus arvensis), chicory(Chichorium intybus), white clover(Trifolium repens), red clover (Trifolium pratense) aster (Aster spp.)and goldenrod (Solidago spp.).2 lb/A Triclopyr plus % Dead Milk- Field Aster/ AverageAmmonium Cys- Wild Canada weed Milkweed bind- Clover, Clover, golden- allnitrate teine carrot thistle whorled common weed Chicory white Red rod species__________________________________________________________________________0 0 30.sup.1 30.sup.7 30.sup.2 50.sup.5 0.sup.13 20.sup.4 20.sup.12 100.sup.4 20.sup.6 10.sup.11 20.sup.9 15.sup.10 30.sup.10 30.sup.12 20.sup.15 301 0 30 30 40 80 0 40 20 100 30 10 30 30 40 30 20 350 1 35 30 30 70 20 55 30 100 30 20 35 20 40 40 20 400 1 40 40 50 65 20* 80 100 100 45 25 40 25 50 50 25** 531 -- 40 40 60 80 30* 100 100 100 50 30 30 30 50 50 30** 535 lb/Atriclopyr__________________________________________________________________________ .sup.1 Applied 7/29/94. Evaluated 8/20/94. .sup.2 Applied 8/1/94. Evaluated 8/20/94. .sup.3 Applied 8/3/94. Evaluated 8/20/94. .sup.4 Applied 8/3/94. Evaluated 9/8/94. .sup.5 Applied 8/4/94. Evaluated 8/16/94. .sup.6 Applied 8/6/94. Evaluated 8/20/94. .sup.7 Applied 8/8/94. Evaluated 8/20/94. .sup.8 Applied 8/9/94. Evaluated 9/12/94. .sup.9 Applied 8/10/94. Evaluated 8/20/94. .sup.10 Applied 8/11/94. Evaluated 9/3/94. .sup.11 Applied 8/15/94. Evaluated 9/3/94. .sup.12 Applied 8/17/94. Evaluated 9/3/94. .sup.13 Applied 8/19/94. Evaluated 9/3/94. .sup.14 Applied 9/22/94. Evaluated 9/3/94. .sup.15 Applied 9/24/94. Evaluated 10/9/94. *100% dead 9/14/94. **Resistant, not killed.
In addition, a series of date and rate tests were conducted between Aug. 9, 1994 and Aug. 25, 1994. A mixture of 2 lb/A triclopyr, 1 lb/A ammonium nitrate and 0.05 lb/A cysteine was applied at 1/2.times., 1.times., 1.5.times., 2.times., 2.5.times. and 3.times. on August 9, August 11, August 16, August 18, August 23 and August 25. Species treated included common milkweed (Asclepias syriaca), field bindweed (Convolvulus arvensis), ground cherry (Physalis heterophylla), dogbane (Apocynum cannabinum), wild carrot (Daucus carota), red clover (Trifolium pratense), whorled milkweed (Asclepius verticillata), canada thistle (Cirsicum arvense), evening primrose (Oenothera biennis), wild lettuce (Latuca canadensis), chicory (Chicorium intybus), dandelion (Taraxacum officinale), buckthorn plantain (Plantago lanceolata), common plantain (Plantago major), creeping charlie (Glecoma hederacea) and poison ivy (Rhus radicans). Complete control of all species was attained at the 2.times. rate of application (4 lb/A triclopyr plus 2 lb/A ammonium nitrate plus 0.1 lb/A cysteine).
It is evident from the above results that the use of thiol compounds in conjunction with auxin like compounds as herbicides greatly enhances the efficiency of cytotoxicity of the herbicide. Substantially lower rates of application can be employed than previously employed to obtain substantially complete eradication of weeds and other undesired vegetation.
II. Use of Thiol Compounds as Root Formation Enhancers
The following study is directed to rooting of microcuttings produced from callus, using auxin shock, followed by treatment with a thiol compound pf malus, Daphne and Dipladnia.
Materials and Methods
Rooting of microcuttings produced from callus of Malus domestica Borkh., Dipladenia and Daphne odorata varigata was studied. All three species were rooted using the same technique. Shoots (2 cm) were harvested at the end of the multiplication medium and were placed for 1 h (Dipladenia) or 2 h (Malus and Daphne) on a highly concentrated agarised solution of IAA at 1 mM (Dipladenia and Daphne) or 2 mM IAA (Malus). After this auxin treatment, the micro cuttings were transferred to a hormone-free medium, containing the thiol compound, dithiothreitol, at different molarities, to allow root formation to proceed.
The plants were first placed in the dark for 6 days at 26.degree. C. and then transferred to the culture room. The auxin (IAA) was solubilized in 70% alcohol and the pH adjusted to 5.5. The auxin treatment took place in plastic jars and rooting was in glass tubes closed with Magenta caps. The actual multiplication and root expression media varied according to the species involved.
1) Malus: For the trials, we used two apple rootstocks (Jork 9 and Cepiland) which were cultured in vitro. The proliferation phase took place on a Murashige and Skoog (1962) (Need citation) mineral solution with 100 mg/l inositol, 1 mg/l thiamine-HCL, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine-HCl, the plant growth regulators benzyladenine (1 mg/l) and indole-butyric acid (0.1 mg/l), and sucrose (30 g/l) (MSO medium). The pH was adjusted to 5.5. The culture conditions were: temperature 20.degree.-22.degree. C., photoperiod 16 h and light (mol/m2/s). To further propagate the plants, 0.5 cm apical shoots were excised and transferred to a fresh medium. After 6 weeks, tufts of shoots between 1 to 3 cm in length were formed.
2) Daphne: An in vitro culture of Daphne odorata varigata was used. The multiplication medium was a Woody Plant Medium (WPMO) mineral solution (Lloyd and McCown 1981) (Need citation) supplemented with vitamins (Mullin et al. 1974) (Need citation) and amino acids (Skoog 1944) (Need citation). The plant growth regulators were benzylaminopurine 0.5 mg/l and .alpha.-naphthylacetic acid 0.01 mg/l. Sucrose and Difco bactoagar were added at 20 g/l and 9 g/l respectively, after adjusting the pH to 5.5.
3) Dipladenia: An in vitro culture of Dipladenia was used. The multiplication medium was based on Murashige and Skoog (1962) mineral, vitamin and amino acid solutions supplemented with 4 mg/l 2iP (2-isopentyladenine) and 0.2 mg/l indolebutyric acid. Sucrose and agar (Sigma 9915) at 30 g/l and 7 g/l respectively were added after adjusting the pH to 5.7. The root expression media were identical to the multiplication media but were hormone free (i.e. a MSO medium for Malus and Dipladenia, and a WPMO medium for Daphne).
The auxin shock utilized indole-3-acetic acid (IAA). The short treatment with auxin was a 2 h dip of the base of the cutting (2-3 mm) in an agarised solution of 1 or 2mM IAA. The shoots were then transferred (rinsed) directly to agar medium for rooting.
With Malus, to investigate the timing of dithiothreitol action on rooting, one experiment was with different incubation timings. One application was during the induction phase (during 5 days following the auxin shock), then the cuttings were transferred to MSO medium. Another application was during the expression phase (after 5 days on MSO medium), then the cuttings were transferred to the medium with various DTT concentrations (day 5 to day 21).
In woody plant experiments, each treatment consisted of 22 or 33 cuttings. Experiments were repeated 2 times. Rooting was measured as the number of roots per cutting after 2 and 3 weeks following the auxin shock for Malus domestica var Jork 9 (easy to root) and Malus domestica var Cepiland (difficult to root) and 3 and 4 weeks following the auxin shock for Daphne odora (difficult to root) and 4, 6, and 8 weeks for Dipladenia (difficult to root).
Statistical data are expressed using GBStat software one, two or three way analysis of variance and means comparison by Dunnett Test at 0.05. The test compares the control treatment (DTT=0). Data are presented as the probability level for each treatment and as the results of the Dunnett's Test.
RESULTS
Daphne odora
Daphne odorata has always been considered as a difficult-to-root species. In our experiments, rooting was greatly improved by using DTT at low concentration (0.1 to 0.25 mM) (Table 10) after 4 weeks following auxin shock.
Another aspect of using DTT is the absence of callus formation at the base of the microcuttings. Large callus are always developing in multiplication medium of rooting without DTT but not in its presence.
Dipladenia
In these experiments, cuttings were obtained either from apical shoot segments (apex) or from mononodal shoot segments. After 8 weeks, the rooting was highly improved using apex cuttings, and after auxin shock, incorporating DTT in the rooting medium at concentrations of 0.1 or 0.25mM (Table 11). As in the Daphne experiments, we also noticed the absence of callus formation in plants treated with DTT.
Malus domestica var Jork 9
With this "easy-to-root" rootstock strain, use of DTT consistently promoted adventitious rooting. Best results were at concentrations of 0.35 and 0.3mM of DTT (Table 12). After 3 weeks, the cuttings without auxin shock did not give a significant number of roots either in the presence of in the absence of DTT.
Malus domestica var Cepiland
This strain of Malus rootstock is more difficult to root than Jork 9. At 3 weeks after auxin shock, the application of DTT under the same conditions as Jork 9 always gave results significantly better than the controls without DTT. The absence of an auxin shock did not induce a significant number of roots (Table 13). Higher concentrations of DTT compared with Jork 9 were required to give the best results in the preparation of plants producing roots and in the mean number of roots/plants.
TABLE 11______________________________________Rooting of Daphne odora after auxin shock in thepresence of varying concentrations of DTT. Data are expressedas % of rooting and number of roots and mean comparison byDunnett Test at 0.05. Means with the same letter are notdifferent from the control (p < 0.001).DTT, mM Rooting, % Roots/plant______________________________________0.00 (control) .sup. 18.sup.a 0.21.sup.a0.1 63 2.700.25 54 1.420.50 .sup. 30.sup.a 0.64______________________________________
TABLE 12______________________________________Rooting in Dipladenia apical cuttings. The best rootingresponse was with 0.25 and 0.1 mM dithiothreitol (DTT) after 8weeks. Means with the same letter are not differentfrom the control (p < 0.001).Type of Auxin DTT,cutting shock mM Roots/plant %______________________________________Apex - 0.00 1.6.sup.a 13.sup.a 0.1 1.9.sup.a 15.sup.a 0.25 2.3.sup.a 15.sup.a + 0.00 3.3.sup.a 30.sup.a 0.1 4.3.sup.a 77 0.25 6.5.sup.a 89Node - 0.0 0.0 0 0.1 0.8 9 0.25 1.1 10.sup.a + 0.00 0.3 2 0.1 0.3 5 0.25 1.1 9______________________________________
TABLE 13______________________________________Response of cuttings of Malus domestica var Jork93 weeks after auxin shock. Means with the same letter arenot different from the control.DTT, mM Rooting, % Roots/plant______________________________________0.0 .sup. 73.sup.a 3.45.sup.a0.1 .sup. 77.sup.a 3.77.sup.a0.15 91 3.77.sup.a0.2 .sup. 82.sup.a 4.090.25 91 4.950.3 96 6.140.5 .sup. 77.sup.a 3.23.sup.a1.0 .sup. 73.sup.a 2.55.sup.a2.0 23 0.23______________________________________
TABLE 14______________________________________Effect of DTT concentration on rooting of Malus domesticavar Cepiland cuttings. Means with the same letter are notdifferent from the control (p < 0.05).DTT, mM Rooting, % Roots/plant______________________________________0.0 (control) 77.sup.a 2.59.sup.a0.1 73.sup.a 1.82.sup.a0.25 68.sup.a 1.68.sup.a1.0 91.sup. 4.64______________________________________
In the next study, species Glycine max, Phaseolus aurus and Malus domestica were studied. In addition, the thiol treatment was varied as to the time administered after the auxin shock.
Rooting of Shoots of Soybean and Mungbean
Seed of soybean (Glycine max, var Williams) or mungbean (Phaseolus aureus) were soaked in water for several hours, planted in moist vermiculite and germinated and grown in the greenhouse for about 2 weeks. Shortly after the epicotyl was fully extended but before the expansion of the first trifoliate leaf, shoots were harvested by cutting just above the roots and transferred to shell vials (10 ml) containing 5 ml of aqueous rooting solution. Rooting was continued in the light (fluorescent, 200 mol/m.sup.2 /s, 8 h light period) for 7 to 10 days. All roots were counted on each of 3 to 5 cuttings in 3 replications. Results are means.+-.standard deviations among replicates.
Micropropagation and Rooting of Shoots of Apple in Culture
The plant was an apple rootstock Jork 9 obtained from B. Kunneman (COST, NL-2160 AB LISSE). The plants were already established in vitro before the start of the experiments.
In the proliferation phase, plants were formed in tufts of shoots varying between 1 and 3 cm in length. To multiply the plants, an apex of 0.5 cm was excised and transferred to fresh medium. Apices were cultured in glass jars containing 100 ml of medium and covered with Saran film (PVDC Dow Chemical). The multiplication medium and the culture room conditions were described in Seingre et al. (1991) Acta Hortic. 289: 151-155. The medium contained MS mineral solution (macro and micro elements) with 100 mg/l inositol, 1 mg/l thiamine HCl, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine HCl, the plant growth regulators benzyladenine (1 mg/l) and indolebutyric acid (0.1 mg/l) and sucrose (30 g/l). The pH was 5.5. The culture conditions were: temperature, 20.degree.-22.degree. C., photoperiod, 16 h and light, 15 W/m.sup.2.
For the rooting phase, shoots of 2 cm in length were harvested at the end of the proliferation stage and were given a short treatment with auxin before transfer to a rooting medium (medium identical to the multiplication medium without hormones) to initiate rooting. The sulfhydryl compounds were prepared aseptically by filtration and introduced with the concentrated molten agar rooting medium just before solidification. After transfer, the plants were kept in darkness at 26.degree. C. for 6 days and then returned to the culture room. The auxin treatment and the rooting were in glass jars.
Rooting is expressed as the number of shoots per tuft producing>5 roots. Each treatment was 22 tufts and experiments were repeated 3 times. Rooting was measured as the ratio of the number of roots produced/total number of shoots.
Auxin Shock
The auxin shock utilized indole-3-acetic acid (IAA). The short treatment with auxin was a 2 h dip of the base of the cutting (2-3 mm) in an agarised solution of 2 mM IAA. The shoots were then transferred (rinsed) directly either to agar (apple) or distilled water in shell vials (soy- and mungbean) containing the test substances.
RESULTS
In either the presence and in the absence of auxin, the numbers of roots were about doubled by the addition of the thiol compounds GSH or DTT compared to no auxin shock (-auxin) or the auxin shock alone (+auxin). See Table 15. Both mungbean and soybean showed an optimum curve in the presence of auxin at 0.1 mM for glutathione and 1 mM for dithiothreitol. In the absence of auxin, rooting overall was much less and the optimum was less clear. However in the absence of the auxin shock, rooting appeared to be stimulated at approximately the same concentration as in the presence of 2,4-D especially with soybeans. Low concentrations of dithiothreitol (0.1 to 0.05 mM) appears to inhibit rooting of mungbean shoots in the presence of auxin.
The appearance of the roots for soybean shows striking differences. Roots in the presence of glutathione or dithiothreitol were not only more numerous but generally were longer than the roots of comparable control shoots treated either in the absence or presence of the auxin shock. Additionally, with dithiothreitol and glutathione, roots formed higher on the stem.
In addition to improving the quality of roots formed in the presence of the auxin shock, glutathione and dithiothreitol also improved shoot elongation following the auxin shock (Table 16). With auxin shock alone, shoot growth was reduced by approximately 50%. At optimum concentrations of glutathione and dithiothreitol for rooting, shoot growth in the presence of auxin was restored nearly to levels in the absence of auxin.
With Malus cuttings growing in agar, the thiol compounds were not only effective in rooting but also promoted shoot growth. As with soybean and mungbean, the compounds were effective both in the presence and in the absence of an auxin shock. See Table 17.
TABLE 15______________________________________Rooting of soybeanshoots promoted by glutathione and dithiothreitolin the presence and absence of auxin shock (2 mM IAA, 2h)Results are meansof ten shoots in each of 3 replicatetrials .+-. standard deviations among trials. Roots per shootTreatment -Auxin +Auxin (2 mM)______________________________________None 15 .+-. 3 28 .+-. 1Glutathione (0.25 mM) 22 .+-. 2 45 .+-. 6Dithiohreitol (0.5 mM) 21 .+-. 4 40 .+-. 5______________________________________
TABLE 16______________________________________Shoot length ofsoybean promoted by glutathione and dithiothreitolin the presence and absence of auxin shock (2 mM IAA, 2h).Results are meansof ten shoots in each of 3 replicatetrials .+-. standard deviations among trials. Shoot length cmTreatment -Auxin +Auxin (2 mM)______________________________________None 10 .+-. 2 5 .+-. 1.5Glutathione (0.1 mM) 12 .+-. 3 12 .+-. 2Dithiohreitol (1 mM) 7 .+-. 1 9 .+-. 1______________________________________
TABLE 17______________________________________Rooting of soybeanpromoted by dithiothreitol and effects on rootlength. Results are means of three replicates of 10shoots each .+-. standard deviation among replicates. RootsTreatment Dithiothreitol Number/cutting Length (cm)______________________________________Control None 13 .+-. 1 2.5 .+-. 0.5(water only)Auxin shock None 19 .+-. 3 0.45 .+-. 0.05Auxin shock 0.1 mM 40 .+-. 10 4.1 .+-. 0.5Auxin shock 0.3 mM 53 .+-. 13 3.0 .+-. 0.4______________________________________
TABLE 18______________________________________Response of rooting of Malus var Jork9 to glutathione (GSH), 3 weeks after auxin shock.Means followed by the sameletter are not different from controls (p > 0.001).GSH Rooting, % Roots/Plant, number______________________________________0.0 (Control) 73.sup.a 3.45.sup.a0.1 77.sup.a 3.77.sup.a0.15 91 3.77.sup.a0.2 82.sup.a 4.090.25 91 4.950.3 96 6.140.5 77.sup.a 3.23.sup.a1.0 73.sup.a 2.55.sup.a2.0 23 0.23______________________________________
TABLE 19______________________________________Rooting of Hederacuttings after 1 month promoted by dithiothreitol.Results are mean of replicateddeterminations of 6 to 9 cuttings/treatment .+-.mean averages deviations between the means. Cuttings withTreatment Roots/cutting no Roots, %______________________________________Control (water only) 2 .+-. 1 33Auxin shock 5 .+-. 2 30Auxin shock + 13 .+-. 9 100.1 mM dithiothreitolAuxin shock + 13 .+-. 0 00.3 mM dithiothreitol______________________________________
Alternative Rooting Procedure
The initial step can be reached by a one day auxin shock with indolebutyric acid (25 .mu.M) on stem discs or 2 h IAA shock (1 mM) on shoots. Both treatments are enough for induced formation of roots in Malus Jork 9. Root induction is obtained in the 4-5 days following the auxin shock, root elongation of expression needing 3 to 5 days more. The time of DTT application in the subsequent steps give results that show promoting effects of DTT. In one experiment, with application of DTT at the time of induction or at the time of expression, preliminary results showed that action of DTT was similar for the induction phase as well as the expression phase.
In herbaceous plants, easy-to-root, the response to DTT and GSH is nearly double the amount of roots produced both in the presence or absence of auxin treatment. With woody plants, which are more difficult to root than herbaceous, the best response is given with only the DTT in the presence of auxin or in auxin shock.
III. Effect of Thiol Compounds on Cytotoxicity of Anticancer Compounds With Cancer Cells
The use of cysteine in combination with an an antitumor sulfonylurea LY181984 (N-(p-tolylsulfonyl),N-(p chlorophenyl) urea. The compositions were tested employing HeLa cells in culture. Mammalian cells were grown in 175 cm.sup.2 flakes in minimum essential medium at 37.degree. C. with 10% bovine calf serum (heat inactivated). Cell proliferation was determined by direct cell counts using a hemacytometer. Cysteine at a concentration of 100 .mu.M was without effect on growth of HeLa cells, but in the presence of 100 .mu.M LY181984 was effective in preventing HeLa cell growth. Cysteine was effective in enhancing the activity of 100 .mu.M LY181984 over the concentration range of 10 to 1000 .mu.M (all concentrations equally effective) (Table 19). Concentrations lower than 10 .mu.M were not tested.
With longer periods of growth the combination of LY181984 plus cysteine began to result in programmed cell death (apoptosis) of the treated cells (Table 18). Overall, the effect of the cysteine was to increase the effectiveness of LY181984 by approximately two log orders, i.e. approximately 100 times less LY181984 was required to kill the cells in the presence of 100 .mu.M cysteine, than in its absence.
TABLE 20______________________________________Response of HeLa cells to LY181984and effect of varying concentrations of L-cysteine.LY181984 L-cysteine cells/mm.sup.2______________________________________None None 933100 .mu.M None 408 10 .mu.M 248 100 .mu.M 268 1000 .mu.M 242______________________________________
TABLE 21______________________________________Cell number after168 of HeLa cells treated with 100 .mu.M LY181894 inthe presence or absence of 100 .mu.M L-cysteine.LY181984 L-cysteine cells/mm.sup.2 *______________________________________None None 1987 .+-. 21 100 .mu.M 2066 .+-. 36100 .mu.M 100 .mu.M 395 .+-. 57______________________________________ *Initial cell number 150 cells/mm.sup.2 between 72 and 168 h. The number of control cells increased 3.3 fold whereas those treated with cysteine plus LY181984 decreased by 10%. Remaining cells were largely apoptotic.
It is evident from the above results, that the addition of thiol compounds in conjunction with a variety of cell modulating agents can greatly enhance the effectiveness of the cell modulating agent. Thus, with compounds which encourage cell proliferation such as auxins, where the auxin can serve as a herbicide or rooting formulation, thiol compounds are found to enhance the effectiveness of the auxin for its cell modulating effect. With chemotherapeutic agents, particularly those associated with inhibition of a surface membrane NADH oxidase, the thiol compounds substantially enhance the cytotoxic effect, permitting substantially lower concentrations of the cytotoxic agent, while retaining the desired cytotoxic effect.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Number	Name	Date	Kind
4169720	Schacht et al.	Oct 1979
4396418	Schirmer et al.	Aug 1983

Thiol activation of cytotoxic and auxin-herbicidal agents and root formation stimulation

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