CA2481110A1 - Vitaletheine and use in cell culture and therapy - Google Patents

Abstract

Novel sulfur-containing derivatives of carboxy-amino-amides are provided. The derivatives have utility, inter alia, as tissue culture media supplements to improve cellular phenotypic expression, cell function, cell production, and cell vitality.
The compounds have further utility in the treatment of neoplasia and other diseases or disorders.

Description

v.cr~x~axe ~~ vsa I~ cant cQr.~;~~~~n .~a~pY
GOVERNMENT RIGHTS
This invention was made in the performance of work under grants ~HHi~ 16,795, SAM 10,628, ,~807RR-05583-25, and BRSG
'707RR-05583-25 with the National Institutes of Health, and the United States Government has certain rights therein.
GROUND O, F Ti_i~TNVENTI013 I. Field f th Invention:
The invention provides a novel group of compounds for modulating cellular activities comprising sulfur-containing hydrocarbon dexivatives of aarboxy-amino-amides such as vitale-theine,[N-(2-mercapto-ethane)-3-carboxyamino--propanamide~,also designated [N-[3-(2-mercapto-ethanamino)-3-oxo-3,1 propanediyl)-carbamic acids , herein referxed to as witaletheine modulatorsrt .
More particularly, the compounds comprise novel short-chain carboxylic acid containing thiol peptides, and their aorresporrding disulfides, as well as compounds representing intermediate oxidation states of these thiols such as sulfenie .
acids and sulfenyl halides; tautomers, oligomers, and rearrange-went forms of these compounds; and further derivatives or forms as described hereinafter. The seminal compound is vitaletheine, 0 O . .
«o_C_~_ ~cxz) z-~-~-t~h) z-sx.
The compounds of the invention are characterized by a pronounced biological activity, and are useful, , for improving the phenotypic expression and vitality of cells in culture. In particular, the compounds of the Invention inexsase cellular lifespan, increase cellular bioproductivity, improve cellular function in culture, and adapt resistant cells to culture.
The compounds are furthex useful, x~er alia, for reestablishing the normal phenotypic expression of neoplastic cells ~,~~ vivo and ~ yitro, and far stimulating immunological surveillance for neoplastic cells. In particular, the compounds inhibit tumor growth, inhibit .metastasis of tumor cells, and xegress tu~aors.

The compounds are broadly useful in the clinical treatment of a number of superficially disparate diseases or disorders caused by underlying disruption of particular metabolic pathways, especially those dependent upon the regulation of thiol-dependent enzymes, bath intracellular and membrane-bound, and have demonstrated efficacy in such superficially diverse applications as adaptation of cells such as hepatocytes to culture; promotion of erythropoesis ~n vitro; and the treatment of neoplasia. The compounds have contemplated efficacy, jn' a ia, in the treatment of AIDS, lupus erythematosus, rheumatoid arthritis, atherosclerosis, hypercholestremia, diabetes, cystinosis, and progeria, and as substitute for erythropoetin (FsPO} .
"Phenotypic cell expression" is defixled herein as the Z5 manifestation of an entire range of physical, biochemical and physiological characteristics of an individual cell as determined both genetically and environmentally, in contrast to "genotypic cell expresaion'~, which in the art solely refers to the expres sion of the cell chromosomal seguence. [see, for example, 2o Qorland's IllustratedlMedical Dictionary, z6th Edition, 19?4, W.
H. Saunders, Philadelphia7. Biological activity of the vitale-theine modulators of the invention thus includes modulation of the expression of genetic material of cells in culture as influenced by the condition and environment of each cell, 25 including the age of the cell, the culture oonditions employed, and the presence of optionally added biological effectors.
2. g~aussion of Related Art:
It is well-known that endogenous thials and diaulfides are critical to the fu»ction of a multitude of dependent enzyme 30 systems in the body, including thiol-dependent and disulfide dependent branch-point enzymes controlling access to major metabolic pathways. Glutathione (GSH, gamma-glutamyl-cysteinyl-glycine, an acid tripeptide thiol} is the most abundant thiol in mammalian cells, and an entire regulatory and regenerating system 35 ensures an adequate supply of this reducing agent, which maintains and buffers call thiol/disulfide ratios. Coenzyme (COA) and lipoic acid are preval~nt in aaammalian systems and also regulate dependent enzyme activity. Xenobiotic thiols such as dithiothreitol (DTT, Cleland~s reagents or dfthioerythritol are routinely used experianentally to regulate activity of thiol dependent enzymes.
In response to demand, thiols such as GSli, CoA, and lipoic acid can, for exa~aple, activate thiol-activatable enzyme by reducing inactive oxidized (disulfide) enzyme to the corre-sponding disulfide (GSSG 1n the case of glutathione-GSH) 3o according t~ the following scheme, wherein P is proteins P P ~ SH SH ~ P--P' S
f--- ~~ ~sH
H Fi H H
Inactive Active (Active] pTT (Inactive) oxidized DTT
Activity of thiol-dependent enzymes is a function of the availability of the thiols involved as expressed by the thiol/di-sulfide ratios of their thiol/disulfide redox buffers (upper arrow): interaction is complex, however, and activity is further dependent on additional faotors such as substrate, ambient ions, and type of reducing thiol (membrane-bound enzymes, for example, are resistant to reduction by glutathione,. similarly, activity of disulfide-dependent enxyn~es is a function of the availability of disulfides, as expressed by the thiol/disulfide ratios of their thiol/disulfide redox buffers (lower arrow).
By the above mechanisms, endag~nous thiol/disulfide redox bufferes such as GSFI/GSSG systems central the activity of many critical enzymes; thyroxine monodeiodinase is exemplary of thiol-dependent enzymes. Regulation of the activity of this enzyme by thiol/disulfide buffer controls the induction of a host of important enzymes, including HMG-CoA reductase, the branch-point enzyme far the isoprenoid pathway, which in turn regulates the production of essential isoprenoids such as steroid hormones, dolichol, cholesterol, and ubiquinone and the isoprenylation of proteins. Glycolysis is also controlled by thiol-dependent and disulfide-dependent enzyme systems; phosphofructokinase, for example, is inactivated by pertain disulfides, whereas fructose-1,6-bis-phosphatase with the reverse enayme activity is activated by certain disulfides. Thiol-dependent enaymes also directly and/or indirectly control isoprenaid and oligosaccharide biosythesis and the synthesis and utilization of thyroid hormones.
One mechanism which has beenidentifie.d as participating in the ' v o regulation of thiol/disuflide equilibria is the oxidation of thiol to disulfide catalyzed by microsomal flavin-containing mixed function monoaxygenase (herein referred to as "monooxygenase"). This monooxygenase catalyzes, for example, cysteamine to its corresponding disulfide, cystamine. Monooxyge-nose activity thus appears to be rxitical to the activity of at least some thiol- and disulfide-dependent enaymes in Carboxylic-aoid containing thiols having an atomic spacing between the carboxylic acid moiety and the thial moiety of from 7 carbons and 2 nitrogens to 5 carbons and 2 nitrogens (glutathione) have been shown to be extremely effective inhibi-tore of the monooxygenase, and glutathione for example, is generally believed to control the availability of thiols and disulfides for downstream dependent enzymes at least to some extent by this mechanism.
Thiols, paxticularly glutathione and cysteine or N-acetyl-cysteine, have been demonstrated in ,.y3vo to inhibit ' neoplasia (piln~.7. Med 91f3C1:1225-1305, 1991); tv inhibit replication of HIV in cell cultures (F~.Sc~N~t~, cad. ~ci. USA
$'7(12j:488~-8, 1990); to ba markedly elevated in preneoplastic/
neoplastic hepatocytes ( 0~l,,Cnrcin.2 (3) :149-9, 1.989).; to influence the proliferation of human perysheral blood lymphocytes (RPBh) and T-cells (8.m;~T.Med 91(3C):1905-1445, 1991) to reverse inhibition of lymphocyte DNA by glutamate in cells fxom Hiv-infected patients (IOt.Immunol. 1(4):367-72, 1989); to reduce infectivity of herpes virus in vitro (~ct.a . V~rol . Praais'11 ( 6 ) :
559~63, 1967) ; to suppress HIV expression in monocytes (~~yge.
~~,. Aced. Sci. 88:986-990, 1991); and to by systemically deficient in iilV-infected individuals (8~i,"ol. Chem. Iip,3gpe Seyler x:101-08, 1989 and The 7~anoet ii:i294~97, 1989). Regulation of FIMG-CoA reductase activity by thiols and disulfides is well-known; as noted above, thyroxine monodeiodinase is a thiol-dependent enzyme, and this enzyme controls the induction of HIriG-5 CoA reductase (]9~ar. J. Bio he~:273-278, 1968). Aypercholest-remia and atherosclerosis, leading factors in heart disease, are now clearly linked to HMG-CoA reductase activity, and treatment of these conditions with various regulators of I~iG-CoA reduetase is known. HMG--CoA reductase activity is also linked to neapla~
sic, most recently by evidence of its role in the transformation of cells by activation of Ras protein which regulates oncogene expression ($ v. Enzyrm~x. X8:373-412, 1973; 8,~,~he~. 6~,~ns$
17:875-876, 1989; Sci~er~, 245:379-385, 1989; 3-Aydr~-3-~,g~~hy~,,q~a~3tarvl Coenxy~e A ~d~ctase, Sabrine ed. CRC Press, Ins. , Hoca Raton, FL, USA, pp. 245-257, 1983).
Another carboxylic-acid containing thiol peptide, pEEDCK, has activities similar to vitaletheine. The reduced form, pBEDCIC, prevents proliferation of hematopoietic stem calls from the bone marrow, while the oxidised form (disulfide) ' stimulates proliferation of these cells. Thus, the disulfide can, for exampe, be used to induce production of normal i:umuno-cytes. Like the disulfide of pEEDCK, the vitaletheine tetramer (V4) , eontins four free carboxylic acid groups and both stimulate colony formation from progenitor cells; conversely, vitalethine and pEEDCK have two free carboxyl groups, and both suppress colony formation from progenitor cells. Vitaletheine is, however, considerably more potent than pBEDCK, especially , vivo.
Vitaletheine also has a close,structural relationship 3o to tetramisole and carbalethiazolidine, vitaletheine has the same nueleophilie spacing as the carbalethiazolidines, and is one carbon longer than the spacing of the nucleophiles than tetra-mfsole (optical isomers: levamisole and dextramisole): Vitale-theine, however, is not stericaliy hindered as i.s tetramisole and the carbalethiazolidines, so in addition to being structurally similar to these prior art compounds, has far greater flexibility for adoption to sterically selective receptoxs.

Both tetramisole and the carbalethiazolidinas have demonstrated utility in the treatment of a host of diseases or disorders, including cancer, autaimmune diseases such as lupus nephritis or erythematosis, rheumatic diseases such as rheumatoid arthritis, immune deficiency diseases, parasitic diseases such as leishmania, hypersensitivity, allergy, hypert~~asion; the compounds have utility also as antilustaminics, analgesics, anti-inflammatory agents, anti-ulcer agents, and as psychotropie, seratonergic, dopaminergic, anti-hallucinogenic, anti-schizo-phrenic, and anti-dysthymic agents, ~~n,e_r alia, in neurological disorders (see, e.g., Qpco~l cpr 38:168-181, 1981; JP 82-57,585;
3P 80-142870] EP 49,902; JP 80-142,869] JP 58-110,595j,,,TP 59 88,491; FR 2,574,408; EP 144,101; FR 2,830,635] El' 49,402] JP 80 142,869; JP 57-130,988; EP 348,746; EP 45,251; JP 59-088,491] Jh 63-225,383).
Additional structurally r~lated compounds having known biological efficacy include calchemicins and esperamicins; the active specie in anticancer activity yields a carboxy-amino derivative mare closely related to vitalethine than the original drug.
Hexamethylene-bie-acetamid~ (HMBA) and sulfide- and sulfone-containing isothiocyanates are further exemplary of drugs structurally similar to vitaletheine having biological activity, particularly anticarcenoqenic activity.
Structural relationships between these compounds and vitaletheine modulators is illustrated in FIG. 26.
Cells which are not capable of continuous growth in culture (non-immortal cells or cell lines) are characterized by a predictable lifespan ,~ vitro, broadly divisible into three 3o phases corresponding to growth, maturation, and decline (i.e., senescence?. Cellular senescence is a phenomenon well-recognized in the art, typically characterized, int,~r ells, by a statisti-cally significant lengthening of~the time required for a mature individual sell to reproduce (genexation time), by the elonga-Lion of normal cell growth patterns reflecting the increasing inability of the cell to efficiently incorporate essential energy and material requirements, and by the termination or statistical-ly significant diminution of the cell s biopxoductivity, which is usually optimal at midcycie (maturity). The life spans of many non-immoxtal cells in culture, particularly mammalian cells, frequently varies from only a matter of hours to only several weeks, even under optimal culture conditions. Sudden, premature death of such cultures fs not uncommon. Even so-called immortal cells, such as immortal insect cell lines or mammalian tumor cell lines, tend to lose viability as a function of time in culture, with aorxesponding decline of the cell mass. Further, many cells, suah as mammalian hepatic cells, cannot be presently adapted to long-term culture as a practical matter.
These inherent limitations on cell longevity ~ vitro have important implications for cultures employed in chemical, industrial, and research applications, and are of particular interest in the,j,h vitrb production of mammalian cell products, including recombinant cell products, especially peptides, proteins, and glycoproteins, such as hormones, enzymes, and immunoglobulins, wherein optimum production is typically obtained duxing the pre-senescent phases of the cell s life-cycle. A
variety of methods have been proposed fox maximizing the production and longevity of cells within existing limitations imposed by cell growth patterns= these are primarily directed to the improvement of culture conditions by techniques for the rapid replenishment of nutrients and removal of wastes, such as perfusion and continuous culture procedures, or to biological manipulation of cells, such as hybridization with immortalising cell lines. While such techniques have generally tended to improve bioproduativity in large-scale applications, the improved results are not usually attxibutable to alteration of cell growth patterns. Further, such prior art methods for improving cell bioproductivities have not been broadly applicable to sells considered non-adaptable to culture; the hepatic cells mentioned above, for example, are currently not maintainable i~x vitro under known culture conditions for more than a few hours.
Methods for the biochemical modification of cell growth patterns have also been proposed to improve celA propagation, but most have been predicated nn the use of cell growth factors.

While growth factors as a group generally tend to increase proliferation of cells in culture, cells exposed to these factors also rapidly become exhausted and die, with little or no net gain in cell bioproductivity. Additionally, such growth factors have not been useful in adapting resistant sells t;o culture.
Tt is accordingly desirable to provide compounds which are effective for promoting the viability and propagation of cells in culture, particularly for promoting cell vitality, cell bioproductivity, cell funbtion, and cell longevity, and for adapting resistant cells to culture. Such compounds are potentially useful not only by themselves, but also in combina-tion with other bioeffectors which are known to promote cellular propagation, for their contemplated combined effects, such as stabilization and augmentation of the cell biomass.
cancer (neoplasia) is popularly treated with chemother-apeutics, debulking, and/or radiotherapeutics, the efficacy of which is primarily dependent upon differences in grorwth rates between normal and neoplastic cells. These therapies have proven to be marginally effective. More recent therapies have sought 2o to fortify bodily defenses against developing tumors, for example by enhancing imnunological responses of the body postulated to be bellig~rent to neoplastic cells. duly in part due to the complexity of the immune system, such therapies have mat as yet proven their value. There is circumstantial evidence that NK
(natural killer) cells may be important effector cells against tumor development in the early stages as described in Immunobi-olQgy o~ Natural Killa~ Cells, volumes I and ITe 1986, CRC Press, Inc. , Boca Baton, Florida, USA, incorporated herein by referBrice.
There is also evidence that through adoptive immunotherapy (the remaval, 3;p vitro activation, and return of immunologioally reactive lymphoid cells to the afficted animal) the regression of established tumors in the animal can be mediated as described in Immune Responses to Metastases, volumes I and II, 198?, Boca Baton. Florida, USA, incorporated herein by reference. It is accordingly desirable to provide a method for normalizing cellular function to interrupt underlying mechanisms of cellular transformation to neoplastic cells, and to identify and enhance, either ,'n vivo or in vitro, those biological responses antagonis-tic towards neoplastic sells.

BR.I~F D~SCRIP I~ 02~i OF THN p~WINGS
Figs. ~ and 8 illustrate the effect of vitaletheine modulator on erythropoiesis.
In the following Figures, mice are injected three 5 times per week, and error bars illustrate the standard error of the mean:
Figure 2 illustrates average basal development of Cioudman 8-91 Melanoma in young (top curve) and old (bottom curve) (Balb c X DHA) mice injected with saline;
io Figure 3 illustrates the significance of the stimu-lation in tumor development produced by injections of 10o ng b-alethine/kg mouse (top curve) relative to saline-injected mine (bottom curve);
Ffgure i illustrates the significance of the stimu-15 lotion in tumor development produced by injecstions of i0o pg b-alethine/kg mouse (top curve) relative to mice injected with 30o pg vitalethine/kg body weight;
Figure s illustrates the difference between tumor development in control or saline-injected mie~e (top curve), 20 and vitalethin~-injected mice (bottom curve) at nearly the endpoint dosage for vitalethine to maximize detection of impurities, such as underivatized B-alethine;
8igura 8 illustrates the significance of the differ-ence between tumor development in mice injected with the 25 B-alethine preparation and with the vitalethine preparation at one tenth the relative ratio o! compounds illustrated in Figure 3, and documents the near quantitative conversion of B-alethine to stable vitalethine by the process of Example IV;
Fignrs '7 illustrates the significance of the in-30 crease in tumor development in five mice injected with 140 ng !i-alethine/kg mouse (upper curve, and logn~ pg/kg mouse g 5 in Figure '7) compared to tumor development in five mice injected ' 11 with l0 pg B-alethine/kg mouse (lower curve, and log~lo pg/kg mouse = 1 in figure 7);
Figu7re ss illustrates average tumor development in five saline-injected ~aice (0) and in five mice injected with one of eight logn0 increments of e-alethine concentrations (from 10 pg jkg souse to 200 ug/kg mouse) ;
Figure eb illustrates average tumor development in five saline-injected mice (0) and in five mice injected with both, one of eight logn~ increments of B-alethine concentra-tions (from l0 pg/kg mouse to 100 ~.g/kg mouse) and i ng vital-etheine V,/kg mouse;
Figure 9 illustrates the difference in average tumor development between mice receiving the combined therapy of Figure 8 and the mice receiving ttie therapy of Figure 7, i.e., the net effect of 1 ng vitaletheine V~/kg mour~e in modulating tumor development at differing concentrations of b~alethine after th~ response to B-alethine alone is subtracted;
giguro 30 (a counterclockwise rotation of Figure 9) illustrates B-alethine concentrations optimizing the antitumor 2o activity of 1 ng vitaletheine V~/kg mouse;
Figure ti (a clockwise rotation of Figure 9) il-lustrates the net effect 1 ng vitaletheine V,/kg mouse in preventing the development of tumor in mice receiving differ-ing concentrations of 8-alethine;
8igure 12 illustrates average tumor development in five mice pretreated with saline (0) and in five mace pre-treated with one of tr~elve logp~ increments of vitaletheine V-0 concentrations (1 fgjkg mouse to 100 ;ag/kg mouse);
Fignra 13 illustrates average tumor development in five mice injected with a preparation in which the vital-etheine V, has been removed by filtration, plotted at the original or unfiltered concentration of vitalsatheine V,o' Figure i~ illustrates the differencf: in average tumor development between mice treated with unfiltered vital-s etheine V, and mice treated with a preparation in which the vitaletheine V, has been removed by filtration;
Figure i3 (a counterclockwise rotat:Eon of Figure 14) illustrates an optimal concentration of the filtrate in de-creasing tumor burden (the filtered equivalenit of 1 ng vital-etheine V,/kg mouse), and the propensity for slower growth in older mice observed in Figure 1;
8igure i6 (a clockwise rotation of lFigure 14) illus-trates the net effect of filtering in improving the tumor-retardant properties of the vitaletheine V, preparation at differing concentrations of the original vita:letheine V, prepa-ration;
Bigots i7 illustrates average tumor development in five mice injected with one of three log~~g increments of vitalethine (100 pg to 10 ng vitalethine/kg mouse) relative to five mice injected with saline (0);
Figure to (a clockwise rotation of :figure 17) fur-ther illustrates the necessity for decreasing the concentra-tion of injections of this preparation of vitalethine to 100 pg/kg mouse or less tee obtain antitumor~activity;
gigots 19 further, illustrates 1) an upper therapeu-tic limit for dosages of vitalethine according to the process-es and methods of the invention (100 pg/kg mouse) in the treatment of Cloudman S-91 Melanoma in mice, using polynomial regression analysis and 3 degrees of freedom (bottom curve);
2) theoretical saturation curves for stimulation of tumor development with poss~:ble contaminants or metabolites (upper two curves); and 3) a theoretical saturation curve describing a therapeutic dose of witalethine at low concentrations, followed by classical maturation kinetics of vitalathine being reduced to vitalethein~e and at higher ooncentr~ations polymer-s izing to vitaletheine 'Vd (polynomial regressio~9 analysis and ~l degrees of freedom, middle curve);
Fig$. 20 and 21 illustrate response of Cloudman S-91 melanoma to treatment with vitalethine modulator in a murine model;
figur: 22 illustrates a nine fold increase in the killing efficiency of a human leukocyte preparation containing NK (natural killer) cells in lysing 100% of huanan leukemic cells (R562) upon treatment of tha cells with vitalethine for 6 days;
Figurs 29 illustrates that the stimulation of tumor cell lysis (K562) with the human leukocyte pr~~paration con-taining NK cells is concentration-dependent, saturating or maximal at about 100 ag vitalethineJml cultures medium;
Figure 3~ illustrates weight increae;es reflecting tumor development in anice inoculated with NS-1. myelama (top curve) relative to normal development of mice (middle curve), and to mice inoculated with tumor and treated with the calcium salt of the benzyl derivative (lower curve) according to Example IIa, IX, and X. Bars are standard era~or of the mean;
Figure 2S illustrates the concentration-dependency of the response in Figure 22, and the lack of tumor develop-ment observed at 100 pg benzyl derivative/kg mouse; and FIB. 26 illustrates structural relationships of vitalathine modulator with various known compc>unds.

SUMMARY OFTHE INVENTION
The invention provides a new group of compounds collectively referred to herein as "vitaletheine modulators"e comprising vitaletheine, a free acid or salt of N-(2-mercapto-ethane)-(3-(carboxyamino)-propanamide], also designated [N-[3-(2-mercapto-ethanamino)-3-oxo-3,1-propanediyl]-a:~rbamic acid];
vitalethine, the oxidized (or disulfide) form of this compound;
biologically-active or -activatable rearrangement forms of these compounds and biologically--compatible salts, hydrates, and oligomers thereof. The modulators of the invention further include biologically-active or -activatabla homologs or analogs of vitaletheine or vitalethine and their corresponding rearrange-ment forms, including salts, hydrates, and oligomers thereof.
The compounds of the invention are useful, aster alia, for promoting phenotypic expression and vitality of cells in culture; including, for example, the promotion of increased cellular lifespan in culture, the promotion of :Lncreased cellular bioproductivity in culture, the promotion of improved cellular function in culture, and the adaptation of resistant cells to , 2o culture. This novel class of compounds thus b~coadly promote the ' vitality of cells in culture for a variety of purpo:es, for example, the efficient and long-term in vitro 7?roduction of cell products for commercial yr research purposes, the clinical comparative study of aberrant and normal cells heretofore resistant to culture, the development and production of trans-plant tissue or organs ~n vitro, and, broadly, the culture of cells for previously purely theoretical biomed:Lcal applications.
The modulators appear to function at least in part by providing a stimulus generic to a broad variety of cel7.s which optimizes 3o cellular production and viability, and provides a starting point for a broad range of contemplated biotech:nical, especially biomedical, applications predicated upon effective cell cultures.
The compounds are also useful, inter alia, for promoting phenotypic expression and vitality of cells v vo; including, for example, the promotion of increased cellular lifespan, the promotion of increased cellular bioproducaivity, and the promotion of improved cellular function.
The compounds of the invention are further useful in the clinical treatment of mammals, especially humans, for 5 example, to alleviate the pain or distressi associated with neoplasia, to inhibit tumor metastasis, to inhibit tumor growth, and to regress tumors.
The compounds are broadly useful, inter olio, for pra~uot3ng phenotypic expression of normal and neoplastic cells, to normalizing neoplastic cells, and/or eliminating these cells from the body.
The compounds of the invention thus broadly promote the vitality of cells for a variety of purposes, for example, the efficient and long-term production of cells and cell products 15 for commercial or research purposes, the clinical comparative study of aberrant and normal cells, especially those cells ' heretofore resistant to culture, the development and production of transplant tissue or organs in vitro, and, broadly, the oulture of cells for previously purely theoretical biomedical 2o applications. The modulators appear to function at least in part by providing a stimulus generic to a broad variety of cells which optimizes cellular production and viability, and provides a starting point for a broad range of contemplated biotechnical, especially biomedical, applications including in vivo therapies.

DETAILED I~1ESCF~'PTION Of ',;'t1E INVhI~'rION
1. _T(i0 ~2~b~93dTL~
The compounds of the invention comprise bioloqically active or -activatabla sulfur-containing hydrocarbon derivatives of a carboxy-amino-amide of the Fos~nula I. hereinafter referred to as "vitaletheine modulators" ax "modulators":
s X(x) s)~ ~_x_~2~Q ~~x_~_Qi~s s ye))~P)X.<r~y x(Oj a (I) 9 ~ s ,~ 3~

wherein:
the set of double parentheses bracke~ts~ the portiotn of .
the molecule basting a charge p when z is 1;
the expression t~-(t'=1Ij M- (wherein C is the ~2cj represantae M~ ( C~tj -M°. M'a (G-t~tr) -!!-, or l~- (C°MAl=N-, and -(C=M)-H- (wherein C is the i~SCj represents -(C=Mj-I~I- or .~ , -(C-MAjsN-; wherein 1~ is X, -1, or a direct bond with the proviso that when -(C~Mj-M- is -(C-MAj=N- or the compound is polymeric or internal cyclic or $pirocyclic, A is optionally R; arid H and . M, are as defined below~;eaah R is independently H or a hydrocar- ', bon radical as further defined hexein;
x is a biologically-compatible cation ar cationic complex as further defined herein;
X' is a biologically-compatible ion or ionic complex Z5 ass further defined herein;
M is S, O, N, or NI3; .
M, is S or o with the proviso that H, ie also optionally N or Nli when the compound is polymeric . or int~rnal cyolia or spirocyclic; ' ' Q is CRz or a direct bond;Qi is CRS, CRJCR=, or a direct bond;
Y is o, -~C=o,-R, or a direct bond;
Z~ is a neutral moiety associated with the remainder of the compound of Formula I;

k 1?
a is the absolute value of 'r/(r~-Hp+~s)~ with the proviso that when (r'+p+~s~ is 20, at least one q or q' is zero such that the sum of any charges on th~ remainder of the complex is balancQd by the charges on the ion or ions, 7C or X', or the s ions, X and X~.
m is 0 or a whole integer from tl to +5;
n is l ar 2 when x is 1, and n is i ow 1.5 when z is 2;
p is +1, 0, Or -1;
q and q~ am each independently +1 or zero;
r and rr are each independently a whole integer Prom +1 to +d, or r~ is a whole integer fro~x -Z to -4;
w is o or a whole integer from 1 to 5; ' a is -i or 0;, .
y is 1 to 40;
s is +1 or +x; a~
the compound.of Formula I has a molecular weight of no more than about 10,000 daltons.
8articularly interesting compounds o! the Formula z are ZO those wherein y ie Prom i to about 20, sapecially from about 2 to 10; or wherein the number average molecular weight of the cxmnpound is no more than about S,OOO daltons, or both; and especially wherein the molecular weight of the compound is at least about 130 daltons.
Pr4ferred compaurWs according to Formula x are compounds o! the Formula II, herein referred to ors "vitaletheine compounds":
-,p_~-~_~.qt2~G~.NlLCS3~CgZ..E.E..S~t Y')~cP~x. ~r'~Z~~~ a (II) ~~ , q JJ Y
wherein R, X, X~, Y, ~, a, ~, n, p, q'', r, r~, w, y, and z are as defined in Formula I.
The vitaletheine co~spounde o! the invention include compounds of the Formula TT in dirulfida farms, camgrising homologous or heterologous (mixed) disulfides; tarisullide forms, comprising homologous o:~ heterologous trisulfides; and oxidized l8 forms (~O) of the homologous or heterologous disulfides or trisulfides, wherein z is 2 and n is 7, or 1.5 according to Formula IIa:
X(s~ ~~yo~C~tJH-Ctt2~CZl~~-tai-CRZ~CR2~s~Z Ym r/2 y (lIa) a wherein R, X, Y, n, m, r and y are as defined in Formula I.
The vitaletheine coaapounds of the invention further include compounds of the Formula I% in reduced and oxidised foe-rns wherein ast, according to Foxwaula IIb:
x~=) (-)~ ~_~_~ ~ ,f~ _G~N~i.~CA.Z-Clt2-Ef-$~ o'~ ~p)Xr (s' )Z~0? i tlTb) -2 2 q a Y
wherein R, X, X~, Y, Z, a, n, m, p, q', r, r~, w, and y are as -defined in Formula II. Particularly contemplated radicals -~-SpYm))~ comprise thioesters and ionised residues of sulfoxy or S-thiosulfoxy acids, especially sulfanic, sulfinic, or sulfonic acids; and when n~~2, ioni:ed residues of thiosulfenic, thiosulfoxylic, thiosulturous, or thioaulfurio acids. Exempir~ry .
radicals -~-~S,Y") ) ~~ inoluds -BOXY (sultanate) , -SX~ (thioiatej , -sI (sulfenyl iodidej, -SI3 (sulfenyl periodids), S~43X' (thiosul-fate); especially SH (thiol or sulfhydryl) and SOFI (sulfenic acid). Aø exemplified above for sulfenyl periodids, a molecule such as Ix or ti~o, or other neutral moiety may be associated with --~(-S,Ys))~'~X'~ or the entire monomer as Z~.
Z5 The modulators of the presetrt invention include -.
biologically-active or -activatabla salt9,, hydrates, chelates, tautomers, oligomers, and rearrangement forms of the compounds cf formulas I, IIae and IIb, and the corresponding salts, hydrates and chelates of these rearrangement foz°avs. The rearrangement forms of the compounds are primarily internal 5 or 6-membered cyclization products resulting from nucleophilic attack on susceptible atoms including oxidised sulfur and doubly-bonded carbon atoms arising from the tautomeriam of the compounds as illustrated in the following Formula Ilce is o.A o.~A .
X(r) (-)p_ ~ ~~ _~ .X.~ -CR -E~-S Y ))(P)X. (r° )Z(Q1) a (IIc) ..
2 2 2 2 , ay~s m q' ..11 y , wherein R, X, X', Y, Z, a, n, m, p, q', r, r' w, y, and z are as defined in Formula II; A is R, -t, a direct bond, or X; and either or bath of the doubly bonded carbon atoms (2,5) are in the illustrated tautomeric form.
Compounds of the formulas T or I=, wherein one or more of the atoms C, H, N, or ~ arc xendered nucleophilic, are readily produced ~,p viyo and ,yR yj ro where they tend to form internal cyclization products, typically stabilized by hydrogen bonds (including hydrates), ions (salts or chelates), or both. These cyclic compounds include apparently biologically-inactive but -activatable "storage°° forms of compounds of the Formula T or II, , which are easily rearranged to the corresponding active com-pound. Compounds of the i~ormulas I and xa and subformulas thereof are fiypica~ly internally cyclized through s or Y, wherein p is zero, or through M,-(C=Mj-H- or -(C=M)-M-, a~a illustrated in , the Formulas Ian and Ib' and following formulas:

o:.,r..=..-... ~ ~. r ... .. ~ .. ..~ ~. .. ... ... ~
~~ P or.. ~. w ~ a- ~.. w. ... r ~.. ~.,.~.~. ~~:r. e. f ' c~ ~~t~a~a ~ vi llrhiuoldins s' ~ j j'/ ~ ~ 1 s~~ '~~T.~~~ i O I
7tQ:)~~sllcl~S.~, ~~.Q~I.p.~-Q~~~s ~_~ r~ , i Itla~) aplsocsaito iepisoeyalie ~ I
I
wtathano ~ r~sifae~se. ( 1 ~1 ~liaossr 1 ~ .
I 4~
.1 le-S~ebiosultoay acid aatas° ' !
(Q ~dis.cC bond and u~Z) l ~s~~w~Aw~, ~
i~
~ ~ rae~alfo%y acid siCtr i ~ hard v0) , ~ ' ~ i ~I
a-wnduns. , ~sbiaso~idi~s '' t~l"s~ ea~o) ~ I ! 1 1 ~ i1 / y v ~e f I~ ~ 11 te) is) ~~~ ,~''~ t~ I f~ tp) ts') ta) ~
x ~ y x. .~.~~'-h-~~Qi-tfsaY~ xv. ~ ~ . i cib. ~
q ' w t '~~ ,e"w~~w I ~ '° ( 1 ,.. ~ ~.....-1 ~ ~. s ~ !
,~L ~"-,r.' L.~..........~....I~..r..
spirocycltc i'~e~~=~Ya=ic sulfoxp acid aster ~
o~ur.~. ~ j v, , ' ~/ .
1 .r ,'' ~r.r r mw w~~ w .r r ~' t spiroc~reiic ehtwu~s.xr acid astas (when n~~ and Q~~dlrect boad)~
i ., ~ , ~atiaossx wherein, in the Formulas Ia' and Ib', M, M" Q, Q~, R' X, X', Y, 8, n, m, p, Cj, ~~, r, ray 6, w, and Z are a8 defined in Formula I; and "c" denotes cyclization.
In general, to form a cyclic urethane of a compound of the Formula I, the charges) on the left terminal nucleophile M, . -(a) moves to the other nucleophile M (9) , either of which may attack the doubly-bonded carbon (5) in the middle. of the ' molecule. Tha developing charge on the central nucleaphile M (6) then picky up an R or X group to form a urethane, or goes on to attack an oxidized sulfur atom, thereby forming a spirocyclic ~. .
urethane by displacing S as illustrated in Formula Ian; or by displacing S or Y, and X' or Z or both X' and Z as illustrated in Formula ~b~; in all oases z or n or both are i after cyclf-ration of the compound. In a similar fashion, the central doubly-bonded carbon (5) can be attacked by one of the nucleo- ' philic atoms S or Y (Formula Ib'), to produce a thiazolidine, or a sulfoxy or thiosulfoxy acid ester, respectively. In this latter case, a spiracyclic urethane is produced when the resulting charge on the central nucleophile (6) attacks the left ao terminal doubly-bonded carbon atom (2) resulting in the displace-ment of, for example, Hzo, IisB, or l~lFi~ from the structure.
Similarly, the charge or developing charge on either a central or terminal nucleophile (atoms 3 or 6, respectively) permits attack upon another monomer of the Formula I to form a dimer, which in turn is capable of polymerization to an oligomer, as described below.
Compounds of the Formula II, including the subformulas thereof, are referred to herein as "vitaletheine compounds". 'The reference compound, herein referred to ae "vitaletheine", and its oxidized form, herein referred to as "vitalethine", are believed .
to be the primary biologically-active forms of these compounds. .
Oligomers of vitaletheine containing from about 2 to about 20 monomers, preferably from about 2 to about 10 monomers, and especially from about 2 to 4 monomers are of particular interest, particularly for their stability. Vitalethine is characterized by the structural Formula IId:

O O
xCr) ~~-)a"c_~_~? ,C&2_C_pTti_Cg,Z-cR2-~~~ r~~ y CIId) wherein R, X, r, and y are as defined in Formula II. Particularly interesting compounds of the Formula IId are those wherein R is H, and X is Zn+s, Ca;~, (CaI)'', (CaOH)'', or other cationic complex. The cationic groups and the hydrogen bonding illustrated in thg following Formula IId' for vitalethine (y~1) appear to add overall structural stability to the otherwise labile aarboxya~aino bone ~~~0-~t c-H .
.~ ~~
..o . . .
. Y
(Iid') iS . . .
u...d ~ s i_'°-i - ~ ~~-~ .
CDctt.a liua iudiG.:t. hrdsogn boaais~g) Disulfides, sulfsnic acids, and sulfenates of Fax~aula I are readily reduced to the corresponding free~thiols, particu-larly in reactions catalyzed by endogenous enzymes, especially reductases and thiol-disulfide isomerases; in particular, vitalethine (Formula Iid~) is readily reduced to vitaletheine (Formula IIe. wherein~R is H and y is 1)t t ~°_~_~-~-~-~_~'~1-~2'sg r CIIe) 3' wherein R, X, r, and y in Bormulas IId, IId~, and IIe are as defined in Formula xx. Exemplary preferred rations X include Zn*s, Ca'~~, or a cationic complex such as (CaI) + or (Ca0li) t, especially Zn*a. Fartiaularly interesting compounds include oligomers wherein y is from 2 to about 10, especially from 2 to 4, and, more especially, also wherein R is Tt. Oligomers of the a~
compound of the Formula IIe wherein y is 4 appear to have great biological potency; such oligomers are referred to herefn as vitalethsins V" Which refers to compounds of the Formula IIe wherein y is 4, and more particularly refers to compounds o: the S Forwula Ize wherein y is 4, R is Ft, and X is a calcium or xinc eation, or a cationic comglex, as discussed in more detail below.
Exemplary biologically-activatable forms of compounds of the Formula II, whSch nay be activatable ~ v~vo or .f11 vitro or converted to vitaietheine of the Formula IId or Iie, include:
1) a disulfide of a cyclic urethane of Formula IIf:
o ~ , , . . ..
'.
O~C-1~-C~,Z~CR~-C-R!i-CB.Z C82-S) s F tilt) This compound appears to be stabilized as a chelate according to the following model:
'~-~..
a o . ~ : ,_' :''" ,~
..., t: ' :_''. :: . .
..i.:'vo~
y .
(Dotted lf~~ tnditlu tome s! I~yase=w iW dins) wherein R, X, and y are as defined in Formula II, especially wherein X is Mg''z and wherein the chelats fs an 1Mq(OH)~ chelate;
2) a dehydrate of compound IIf, comprising a cyclic urethane imine of the Formula IZf':

. :.
wherein R is as dstined in Formula II;
3) a hydroxythiazolidine of the hormula IIg:
0-a ) (' ) . ~_ .. . . . _ ' .
X O ~ lOi CRZ C~, C-b~t~CR3 CB2 8 ~ y (IIg) wherein x, R, y, and r era as defined in formula II and A is R, l0 X, a direct band, or -1 as defined in i°oriaula IIc;
~~ a thiazolina of the Formula IIg', in which 8ormula Ixg ie dehydrated to the thiazoline in a manner similar to the dehydra-tion of compounds of the Poriaula IIf to compounds of the Formula IIt' : ~ . .
X(r) <')0 ~-~_ .~2-~.CRZ-CAZ-5 r y (IIg~) ...
..
wherein X, R, r, and y are as defined in Formula II;
5) an ionized hydroxythiazolldina of the Formula IIh, as follows: _ ' ,~ , .
O(.) ' .'.
z~ ~ I
X(r) ~(')0-~,.~"~_Cgl-C-l~li-CRZ-CRZ-SJ rPZ y (IIh) .
wherein R, X, r, and y ars as defined in Formula II; ar forms of the thiazolidine of Formula IIh 1n which the cyalization propagates through the carboxy-amino moiety as in Ia' to form:
a5 a) intermediates of the Formula IIh°:

t-)0 . D . .. .
~ICr) t~) ~ , ~ _~,. .og .g tlIh' ) 0 C DiH-C~Z-CRl ~ r,2 .
which are dehydratable toc 5 b) a spirocyclic urethane-thiazolid3ne of the Formula IIi: '.
.. .
C~~ . IIi 0~.~_~2.p~Ø~.~ t ) . .
10 or ~ .. .
c) a~n imidocarboasate tautomer of the Formula Ixi~: .
~0 Xtr) t )0-~-~ -~ ~ ~~~ -s (II1' ) 2 2 ~2-~'1 x y 15 wherein X, R, r, and y in the Formulas Ilhr, YIi, and Ixir are .
as defined in Formula II.
Other potentially activ~atab,e rearrangement forms of vitaletheine include the following:
6) aulfsnatss corresponding to the cyclic urQthanes of the 2D Formulas IIf and II~r of the Formulas IIj and IIj't -.
wc-tai-caZ-cxZ.c.ara-csZ.r.~z-s-ox ~ tlx~) o-c~l~.caz~caZ~c~Di_cXZ_c1~2_s_ox y tts~~) 7) cyolic sulfsnaters corresponding to the thiaxolidines of Formulas IIg, ITh, and iah' of the Formulae IIk, :CIm, and IIm~:
0»d O , , .
Z~r~~~''O-C~.~_CaZ.Cg~_C~t1g_CR~~C&~~gp~ r y . (Ialc7 .~,~."~,~r .
~(.' C Z z ~ z ~=~a ('~0 0 io x(=1 (.) ~ _~.ca _ ~ _~_c .so (zxm') y which are dehydratable to: ' .

8' the corresponding dihydro-oxathiaai~e of F'armuls IIk~:

7t(r~~( ~p'f _~»~~'~°C'~°~Z'ptl'g ~ x y (IIk') i or 9) the corresponding:
a) spirocyclic urethane-~ulfenate of the Formula IIn:

as ' O~C~tiil CRZ~C~2~C~!t!1-CR2~C~Z~8 , (aan) ' b) or the corresponding fmidacarbonate tautomer of Formula IIn~:

(-) _ ~ .CR -Cx -C-llfi-C8 ~CA ~8~0 x p x ~ z s J= y o a~
wherein X, R, r, and y in the Formulae IIj through IIn~ am as defined in Formula II, and A is as defined is Formula IIc; and the various Formulas II further include rearrangement foryas within the scope of the invention as deacriDed herein, particu-larly as described for Formulas Ian and Ib~. .~, The modulators of the invention further comprise biologically -motive and -activatable derivatives of the vitaletheine modulators of the Forsaula I, characterised by the following Formula III, herein referred to as ~"vitaletheine ..
i0 derivatives": - :. .
w g~L)r ~8)~-C$.j-Q ~;;M-CR2~Ql"f~"g~; Y~))t1E)$o(s'.).l(C) i (III) . .
4 ~~ ''~ q '~ ~ _ Y
wherein Mi is S ar O; M is S, O, N, or HH; at least one id, or M
is other than O; and R, ~r SEA X, Xy Y, Z, a, n, lRa pe qr 9'r r~ ~ .
r', s, w, y, and z, are as defined in Formula ~; wherein the dotted li.nea are bond resonances or tautomerisms; and wherein in compounds .of the Formula III which are internal cyclic and spirocyclic compounds, K, is additionally optionally M as depicted in Formulas IV through Vleo.
Particular da~r3vatives within the scope of Formula III
include homologous or mixed E~uifidse, homologous or mixed trisul:Cides, and oxidized forias (m>0) of the homologous or mixed disulfides or trisulfides, wherein z'a and n is 1 or i.5 according to Formula IIIa: ~-.
H x w X(=) ~~-)ttl-C-13-CRI-Q-~-li-CRZ-~Q1.S~2 Ya' x~,~ y (IIIa) wherein x, ME, Q, Q" R, x, X, m, n, r, and y are as defined in Formula III; and X is espeoially If', Zn'"s, calcium ration, or a ..
calcium cationic complex. ' ., Further derivatives within the scope of Formula III
include the reduced and oxidized forms of compounds of Formula III wherein x=l, according to the Formula IIIb: .

xCi) (-)bl ~_~_~~Q_~_x-~~~1~,~,Sri s» (P3x~(r')~(0)~ a (IIIb) -_ t q w Y
Wherein M, Mfr Qi Q~e ~e Xr X°r y, $, a, IOr nr pr Qrs rr rn wr and y are as defined fn Formula III, and X 3e especially H*, Zn*sr calcium ration, or a calcium cationic complex.
the compounds of the Formula IiI also include these compounds in the foam of their biologically-active or -activata-bIe tautomers, clhelates, hydrates, and biologically-compatible salts as described for Formulas I and II, and rearrangement products thereofr including .compounds based on nueleophilic cyclization according to Formulas Iar and Ib'; and 'further include tautomsric derivatives of compounds of the Formula III
as described for Formula IIc$ as summarized in Formula IIIc:
is ~'~ x~'~
x(r) ( )H - ~ -~ -Q- ~ -08 ~Q -~-S Y ~)(p,X'(r~)X(0) 4ITIe) .
1 2 2 1 ~z se q, v s .

Wherein 14, Mlr Qr .~yr Rr Xr $y 'iCr Z, a, m, n, p, tj', r~ ry w, ,' .
y, and z are as defined in Formula III, A ins as defined in Formula IIc, and either or both doubly bonded carbon atoms (2,5) are in the illustrated tautomeric form.
Additional compounds within the scope of the invention include modulators of the Formulas IV-VIr and the subformulas thereof, wherein Mi in the compounds of the Formula I is M:
H M-A . , r ~ w ~-~-~-a-~-H-~~-Ql-s~2 y~, y (IV) wherein M, Q, Qir Rr Y. m, n, and y are as defined in Formula I
and A is as defined in Formula IIc~
Further compounds of the present invention comprise biologically-active and activatable compounds o! the Formula V:

H~ , ' r t . .
~M-C-!!~CRZ~Q-C"Ny ~~Ql.gA~~ Ym (V) .
wherein M, a, R~, R, Y, rn and n are as defined in Formula Z. ~ ' The compounds of the present invention further include biologically -active arui -activatable forms of compounds of the Formulas VI and the following thereof in reduced and oxidized forms, which comprises - . ~:
1) cyclic urethanes a! the Formula vI:
'a . .
-c-H~c~-Q-a-H_c~-Ql,~-s~z ya7y(r7x.(=')tto7 {vI7 .
J' wherein the urethanes are substituted as defined in Formulas Tlgr I Ig, and I Ih; !d, Q, Qt, li, 7t, R° r Y, Z, m° y P' 9 ° , r ° , w r Y r and s are as defined in Formula I, and A is as defined in Formula IV;
Z) cyclic iirines of the Fonaula vIa comprising urethanes dehydrated as analogously illustrated in irormulas IIf ' and IIf°: .
1t~ : : ._ R. ~ .~ .R:r,.p- .Q -~-g~ Y 7)tP)x.(r )~(~) (VIa) 2 ~ 1 ojx m , l~her6in M, R, Q1, R, SCE, Y° Z, n, m° p, Q° r r', w, Y, dnd Z ari8 as defined in Formula I;
3) spiracyclic compounds of the Far:aulas VIb and VIc analogous to preoursars of the spirocyclic urethanes of the Formulas IIh°and IIn:

so (')x M
x(r) (-)!i-C-M-CR - ~ ~M- -Q °~E-S Y )){P)x~~r')x(0) (VTb) Z ~2 1 n at q, x r/2 (~?~ ~ 'M ~.
x(r) . ., .Q --~-s Y )'tpy.err)~(o) ~~) .
~2 ~2 1 n m q, x t/Z
Whar6in H, y fit, R, $~ X°y Y, ?y 17, fi, p, ~j~s r, rr~ W, alld Z
~ .
i.o ara as defined in Formula I;
4) corretapanding spisaayolia urethane-sulfoxy (trn~~i) or urethane-thiosulfaxx (n~2) acid esters (Formula VId), or .
uretha»e-sulfides (Formula VIe), respectively, formed by elimination of sulfide, nitride, nr oxide from the oampounds of the Forrulas VIb arid VIc as HZS, H3H, ar FisO:
..
.H. .Q ..qt..s y )'~p)xr (r' > f(0) vxa . . ..
2 ~2 1 rt :a ( ) , .
g~ v ~M . . . ..
(p) dxr) (o) M-C-M~CMZ-Q~C-M-CRS-Ql-(~fr-S~Ys)) )CQ~ ' Y . (VIe) :err Wh~refl8 M, (,Z, Rlr Rr X~ r yr ~r mr ~r W ~t s r~ r ~ w ~lre defillAd .
as in Formula I; or 5) imidocarbonate tautomars of compounds of the Formulas VId or VIe, as describad for Formula IIi':

. ~ , x(r~ ~( )5.~"F.~ga.q.d_H.~l~Qi.,~",guya)y(P)X.(r ~I(a)~ r (VId') , ..
q w y , --....~ .
/~M
y(r) ('~x.~/../p.~ . ~ .X. .Q .~.E.S Y )~cP)y(r')Z(03 (vxe') 2 ~2 1 a a q, w r , y fidherein M, W Q!r ~a xt Xrr Yt Za nr mt pr q~t rsa wr ye and Z
l0 are as degined in Formula I.
The modulators of the present invention especially include biologically-active or -activatable salts, hydrates, chelates, !automate, and rearrangement forms of oligomers of avonwaers of the For~rula I, particularly oligomers of monomers of ' 15 the Formula IId, herein referred to as witaletheine oligeaaers~, comprising polymerization products of ~ronomers of the Formula I ' and subtox-~sulas thereof, incltuiing cyclizations according to Formulas Ia' and Ib~l, and the cotta~ponding salts, hydrates, !automate, and chelates of these !ores. Oligomers produced by 2o tho polymerization exemplified in Formulas zap and Ib~ appear to be reaistarit to rearrangement and provide storage farms of ..
compounds of the invention, which, however, may still be labile to certain organic solvents such as ethers and nlcohols.
Hreforred oligomers of monomers o! the Formula I and subformulas 25 thereof are those whexein y is lrom about 2 to 1U. Particularly useful preparstlons of vitaletheine, include those prepared, !or example, according to Example III, esp~cfally those oomprising a vitaletheine ollgomer of 4 monomers (y=4 in Formula TIe and .
Formula IX following), and particularly optionally including 3o minor proportions o! at least one other oligomex or compound of the invention. This tetramer and vita3ethine appear to be particularly active. Formation of this oligomer (herein referred to as ~tV~~) appears to occur through an initial nucleophilic attack o! a first monomer on one of the doubly-bonded carbons 35 (2,5) of a second monomer to generate a nucleophilic oxygen from the carbonyl oxygen (6) of the second monomer. Polymerization of the monomers of Fonaula I and the subformulas thereof, for example oligo:aers wherein y is about 30 or less, appears to be propagated through this initial alkaxide ion (the nualeophilic oxygen 6 resulting from the initial dimerization) until the polymer folds back an itself and the last alkoxide ion present tthe fourth in the case of V,) reacts with the first 4initiatingy monomer. An intermediate dimer, exemplified it: Formula VII, is comparable to a benzyl derivative of Formula VI7tI, obtained as 1o a by-product under certain conditions (sea, e.g., 8xample CIA) in the synthesis of vitaletheine v,,:
=<r) (vx3) stt (Cs30i1t$)~ ~ ~ p....p p,- ~, ~-C.p-C&~ dilil~
-... ~'~ \ '~/~
The monomers alternately era linked by Y when X is the initial Z5 attacking nucleophile, according to Formulas Ia~, Ib~~ and X.
The reaction terminating the polymerization is apparently a nucleophilic substitution of the original nuoleo-phile invoived.in the formation of the first alkoxide ion by the last alkoxide ion, resulting in a cyclic polymer of monomeric 3o subunita, which are nearly identical in speotroe~copic analyses.
once formed, the polymer appears to stabilize the oarboxy-amino moieties through salt bridges within the oligomer, and statically prevents rearrangement to other active or activatable forms.
Vitaletheine Vy (the tetramer of vitaletheine, Formula IIej is 35 illustrated in the following Formula IX:

~o ..

=tea e-ao.~.~".~.~ .,a.,c,~.e~.et-a=,t.,~toa r S . , $tsa t .
tt~tJ
=tra t ' ~ts' t aa,~,ys~~.~~! ~.p~~t'st aa.toaxtCa , ,..,.
r wherel» R, X, x~, 2, r, and w are as pr4viousiy defined in Formula I; preferably X or X~ is a portion of the cation Zn+a having a chsrge of +i and X~ or X, respsativelyr is H*; and 2o especially when X' is a portion o! Zn*' , X is H*, r is +1, Z is I~O, and w is 2. In the preparation o! vitaletheine Vs as described in Exa~rgl8 III; ~ ~1 H* and 2 Zn'a neutralize the amino-carboxylate a»d thiolate charges, and the entire complex contains 8 moles of hydration per ~aoie of complex.
Decomposition or rearrangement of vitaletheine V~ is .
induced by some organic solvents such as sther, and by heating, which apparently results in decarboxylation of the palysner.
Accordingly, caution should bs exercised during purification procedures to obviate loss of product. ' The modulators of the present invention rurther include .
biologically -active and -aativatable derivatives of the vitaletheine oligomers of the following Formula X, wherein a compound of Formula IT1 is polymerized as a monomer via nucleo-philic attt~ck on one of the doubly-bonded carbons (2,5':

3$ ..
x x , w gq )~~~)~".~.M~~~Q.~.M_~3.q1..~.~..g~Z ys))(P)xq(r')T(0)1 $ cx) x J Y
r _ .
wherein the attacking nuchsophile(s) comprises) 111 (a), M (3,s), S, or Y as described for Formulas Ian, Ibs, VII, VIII, and IX, and arise- through the tautomerizations described herein, particularly as described for Formula iIIc; and wherein M, Mi, Q, Qi~ R~ Yi xi x~~ x~ ZW'o ni ~s mr W 9~ ~~i r~i ar wa and y arc as defined in Formula I.
In Compounds of the Formulas I through X, and the various rubformulas thereof, the hydrocarbon radical R is ~' sulc~tftuted or unsubstituted, saturated or unsaturated, with the provisos that compounds within the scope of the invention have a molecular weight of na more than about 10,000 daltons and contain less than about ~0 manomiers (y<a0) ; preferably, campounde according to the present invention have a molecular weight of no ' more than about 5,000 daltons and contain lees than about 20 monomers (y<20j' most preferably, compounds according to the invention have a molecular weight of at least about 130 daltonsf compounds containing from about 2 to 10 monomers are especially interesting. Further, any hydrocarbon substituents R present must not substantially adversely affect the bio~unction of the molecule, either chemically or stereochemicaily.
Preferably, hydrocarbon substituents R comprise Z5 suitable lipophilic moieties which counterbalance the hydrophilic portions of the molecule to promote the transfer of the module-tore of the invention across tho call membrane to maximize intracellular reactions as understood by'thase skilled in tha ' art. Further, R is most preferably selected to avoid etereochem-ical obstruction or biochemical inactivation of the active .
functional groups of the molecule, particularly the carboxyl-terminus and sulfur-terminus moieties which are apparently critical to the biological function of the molecule, both in their chemical constituents and their physical presentation to the cell. The substituents R are thus not critical to the - .
invention, as long as these groups minimally function as described, do not substantially interfere with the biological activity of the molecule, do not substantially promote decomposi-tion or unwanted side reactions of the molecule, either intracel-lularly or extracellularly, and do not substantially render the 5 molecule toxic to the cell; such hydrocarbon radicals R are referred to herein as g'physiologically-acceptable hydrocarbon radicals R".
Exemplary hydrocarbon substituents R are C,-Cm-hydro carbons, especially C,-Cpe-aliphatic or -cycloaliphatic radicals, to which are branched or unbranched, substituted or unsubstituted, saturated or unsaturated, particularly C~-Glr-alkyl or -alkenyl;
ox substituted or unsubstituted mononuclear or polynuclear aryl, especially phenyl. An exhaustive list of potentially suitable hydrocarbon radicals R is set forth in United States Patent 15 4 , 216,160 to Daru~~ et aI . , incorporated hexwire by reference, especially the hydrocarbon radicals R, and R2 described therein.
A particularly suitable substituent R is Ii.
In the compounds of the Formulas I through X, X or X' ' is H+, hydronium, or a cation or an organic or inorganic cationic 20 complex; or X~ is additionally an anion or an organic ox inorganic anionic complex; and each X or X~ is selected for biological compatibility. The nation or cationic eompleuc X is monovalent, divalent, or polyvalent, especially monovalent, divalent, or trivalent wherein r is +1, +2, or +3; the ion or 25 ionic complex X' is monovalent, divalent, or polyvalent, especially monovalent, divalent, or trivalerxt wherein r~ is -3 to -1 or +1 to +3. ~C or X' each comprises an ion or ionic complex which doss not substantially irreversibly inactivate the active portion of the molecule and which doss not substantially 30 interfere With the biofunction of the active remainder of the molecule, either chemically ox stereoehe~nically; such ions or ionic complexes X or X~ are referred to herein as "biologically-compatible ions". Some ions may inactivate the molecule while they are present, but the inactivation is readily reversed, for 35 example spontaneously, enzymatically, or chemically,~ such ions or ionic complexes are within the scope of the invention, as it may be convenient to prepare an inactive molecule and subsequent-iy activate it for use, especially in the preparing of molecules ~ .
targeted for activation and use in specific cells or tissues.
Modulators of the invention in solution are highly sensitive to electrolyte concentrations, and are easily irreversibly inacti voted by excess amounts of compounds or many electrolytes, particularly magnesium ions. further, the ions X and X' may shift an existing equilibrium between a biologically-active form of the modulator and a corresponding storage form of the modulator in favor of the storage form, or y~lce versa. Exemplary cations X which appear to stabilize the molecule in either native or activatable form include Ca*Z, (CaI)+, (CaOH)*, and especially Zn+s, which favor the active form, and ~Ig+z, which may favor an activatable or storage form. Exemplary ions X° include H+, T, periodide (I3-) , Zn+2, or Ca+a. As described herein, a charge >+1 25 on the ion X or X' may be apportioned between two or more .
negative charges s or p on the remainder of the molecule to fare one or more salt bridges within the molecule or between mole-cules; the "ion X "' in this instance accordingly comprises a portion o! the ion X, or vice versa. A positive ion X or X' having a charge greater than *1 may form a bridge between a group bearing a charge of s wherein s is -1 and a group bearing a charge p wherein p is -1 in a given molecule, or between two groups bearing the charge s wherein s is -1, including molecules wherein y~i; or in molecules wherein y>1, they may form a bridge between two groups bearing a negative charge s, or two groups bearing a negative charge p, or between two groups one bearing a negative charge s and the other bearing a negative charge p.
When p is +1, an ion X' having a charge leas than -1 may also form a bridge between two groups bearing.a positive charge in the same molecule. Additionally, an ion X or X' may ohelate two identical or different monomers or oligomers of the Formula I.
Generally, the total charges on the ions X and X° present will balance the total charges s and p on the molecule; however, in some instances, a portion of the total charge on the molecule may be balanced by one or more ions extraneous to the molecule.
In compounds of the Formulas I through X, the neutral moiety Z~,(o) is a neutral molecule or another neutral moiety :..

which is associable with the compound of the Formula I and subformulas thereof as indicated. Exemplary neutral moieties Z"(o) include for example, iodine, H20, polyethylene glycalg, and polyaxyethylene ether detergents.
several inactive but activatable forms of the modula- .
tore within the scope of Formula I have been identified, including those described above, which appear in some instances to be inactive °'storagen farms of the modulators, capable of .fit yiyo ~ stn vitro rearrangement to one or more active forms.
IO v v rearrangement or jn yitro rearrangement fn the presence of living cells appears to be a result of the action of endogenous ' enzymes as mentioned above, which, depending upon the type of cell or cells and culture conditions, may convert inactive forms of the compounds of the invention to the corresponding active form, especially in the case of the vitalethine ax vitaletheine compounds. Proteins and hydrophobic environments such as cell membranes may assoc~.ate with and stabilize the active form of the product. Rearrangeraent of inactive but activatable forms may also be induced by other means as described below.
2o within the present context, '°biologically-active or -activatable" refers to compounds within the scope of Formulas I through x and the subformulas thereof which are biologically active, or which are activatable to biologically active compounds on exposure to activators such as the following: chemicals 2b including biochemicals such as enzymes and selected organic solvents, acids, and bases; radiation including electramagrietic, actinic, ar radioactive energy; or heat energy. Inactive compounds which respond to such treatment to became bioactive are referred to herein as °°activatable" and,are included within the 30 scope of Formulas T through X.
Certain compounds of the invention, and other substanc-es which are postulated to inhibit the degradation or metabolism of the modulators, are useful in aombinatian with the modulators of Formulas I through X. At low concentrations especially, 35 degradation catalyzed by endogenous enaymes represents a mechanism for significant losses of added modulator. Compounds which inhibit these enzymes, Without themselves interfering with the action of the modulators, potentiate the action of the modulator by making sustained, lour, effective concentrations possible.
II. ra~aration of the Compounds:
Compounds according to the present invention, particu-larly compounds of the Formula IIe wherein R is H, are postulated as endogenous to a substantially complete spectrum of plants, animals, and microorganisms, and, accordingly, it is contemplated that the compounds of the invention are recoverable from a variety of organisms and isolatable for use according to methods well-understood in the art. It is further contemplated that the recited biaapplicability of the compounds to the function of the broad spectrum of cells recited below is attributable to the ubiquitous, or near~ubiquitous presence of these compounds in virtually every living sell and the essential presence of these compounds for the autoregulation of cellular life. .However, since the endogenous compounds are thought to be present, ~ .>,rivo, in extremely small amounts, and are known to be easily converted into inactivatable forms, fox example by customary purification 2o methods, it is recommended that the compounds of the invention bs synthesized for use, especially to avoid contamination of the product with ~aitogens, saponins, pathogens, antigens or other potentially reactive compounds present in bioi~ical materials, and to prevent the undesirable rearrangements described above.
B5 At present, the most potent of these compounds appear to be those within the scope of Formula IId, viz., those based v on the bis anionic vitalethine, [N,N'-(dithiodi-2,1-ethanediyl)- , bis-(3-carboxyamino-propan-amide),, also designated as 3,3'-[dithiodi[(2,1-ethanediyl)amino)~-bis[H-(3-o~eo-3,1-propanadiyl)-30 carbamic acid], and poly~era of vitalethaine. Analysis of the w-polymers by filtration through a P-2 gel column indicates that the monomer of vitaletheine (Formula IIe, Wherein y is 1 and R ;
is H) tends to spontaneously polymerize during purification to form rultimers, especially oligomers wherein y is from 2 to 4; , 35 the preparations of the V, oligomer and vitalethine, especially, have extremely high biological activities.

The (13c]-HI3R of vitaletheine Vd (Formula IIe or IX, wherein y is 4 and R is H) indicates nearly homologous subunits;
the tetramer (y=4) is an extremely rigid structure similar to those reported for certain ortho-ester-like compounds in Tetrahedron hatters 22:4365-4368 [1981] (incorporated herein by reference). Based on (1307-tit analysis, the multimeric vitale-theine structures are postulated to be polymers which are formed by the attack of nacleophilic oxygen (6) derived from the central amide on the carbonyl carbon (5) of another monomer, probably through initial attack of sulfur or atom Y according to Formula X on the carbonyl carbon (5) of the amide of the initiating monomer to generate a nucleophilic oxygen (alkoxide ion) from the carbonyl oxygen (6). Polymerization may be propagated through alkoxide ions in a manner Which resembles ortho-ester formation, until the polymer folds back on itself and a terminal aikoxide ion reacts with the original monomer. The polymerization is then terminated by nucleophilic substitution of sulfur or atom Y
according to Pormula X which initiated the polymerization with a terminal alkoxide ion, resulting in a cyclic polymer which a0 typically contains homologous monomer subunits. Slight puckering of the polymerized (-C-o-)Q ring (n is from about 3 to about 24, , usually 3 or 4, especially 4) split observed resonances in the above-described Mgt analysis of V~ inter four minor peaks in the range calcsulated for a highly constrained quaternary carbon atom.
Polymerization of the monomer does not appear to result from manipulation of the monomer by the apglied analytical procedures, since this NMR evidence indicating a tetramer was obtained prior to determination of the molecular weight of the polymer by gel filtration.
jest Modes for Preuaring C~ounds of the Invention Although vitalethine is also prepared by the above -procedure (Examples IIa and IIIa), carboxylation of 8-alethine y by reacting the disulfide with phosgene in the appropriate chemical milieu is the preferred method of synthesis. packing of the reaction vessel in dry ice controls the exothermic reaction and improves yields of large-scale preparations.

Similarities in the physical properties of these two potent biomodulators, i.e. thermal lability and infrared spectra, are described in Bxamples III, IV, and V.
The compounds of the invention yrere conveniently S prepared employing 13-alethine blocked with a protective group such as N,N~-his-carbobenxoxy-- (CBZ-) as starting material. The blocked B-alethine was then selectively debloc~ced by the process of the invention to remove benzyl groups and yield the compounds of the invention. Techniques for the synthesis of the blocked l0 B-alethine staxting material are present in the literature;
however, the known techniques generally provided a product of low yield or purity, or both. Many of the impurities obtained in known procedures result from the combined poor solubility of the product compound and the dicyclohexylurea by-product produced in 15 coupling reactions which utilize dicyclohexylcarbodiimide, According to the process of the present invention, product purity and yield are improved by first coupling CBZ- or y similarly-blocked B-alanine to N-hydroxysuccinimide (commercially available from Aldrich Chemicals, Milwaukee, ~I, USA) to p~oduee 20 the corresponding N-hydroxysuccinimide active ester using dioyclohexyicarbodiimide (commercially available from Srlawars/ .
Mann, Orangeburg, NY, uSA) following the procedure described in J.Am.Chgm.Soc. ,~6_: 1839-1842 (1964), incorporated h~lrein by reference. commercially available starting materials, such as -, 25 N-CBZ-8-alanine (Stigma chemical, St. 7~ou3s, M4, UtSA), are first coupled to N-hydroxysuccinimide (Aldrich Chemicals), with s precipitation or the dicyclohexylurea by-product. The salable active ester product is recxystallized and coupled to the free amino groups of cystamine, readily obtained frota cysteamine ' 30 (available from Aldrich Chemicals) by oxidation with peroxide, for example, by titration in acetanitrile with peroxide until no reducing equivalents are evident. This is conveniently monitored using strips of paper soaked in a solution of O.1M potassium phosphate buffer and lflmM 5,5'-dithiobis-2-nitrobenzoic acid 35 (Sigma Chemical) and dried; residual thibl in the peroxide/
cysteamine mixture produces an intense yellow spot on the paper.
water added with the peroxide and produced as a by-product of cysteamine oxidation is readily removed by repeated evaporation of the acetonitrile azeotrope priar to coupling with the soluble N-hydroxysuccinimide active ester obtained by dicyclohexylcarbo-diimide coupling (aunxa). Using this form of cystamine instead of a hydrochloride or. similar salt ensures more complete reaction of the active ester with the cystamine, since this reaction is dependent upon a nucleophilic attack of the free amines of cystamine on the carbonyl carbon of the active ester. N-hydroxy-succinimida is regenerated as a by-product of this reaction as the blocked !3-alethine precipitates. Tha benz~yi groups are then removed from the blocked B-alethine as described, for example, in Examples III and IV, and the product compounds recovered.
III. ~,F~ilitY o~ the Comgi2u~s:
A. The vitaletheine modulators of the invention are .
I5 useful, in~erwalia, for improving cellular phenotypic expression and cellular vitality, inin vivo or ,~ vitro, including, for example, increasing cellular lifespan in culture, increasing cellular bioproductivity, improving cellular function, and adapting resistant cells to culture, especially for enhancing oellular bioproductivity and tar adapting resistant cells to culture. The processes oP the invention are particularly applicable to those cells not capable of continuous growth under conventional cuiture conditions, especially "normal" mammalian cells. As defined herein, "normal" cells comprise non-transform-ed, especially non-virus transformed or non-tumor transfoxyned cells, including non-transformed sells ~rhich are functioning abnonaaily in some respect, such as cells wherein bioproduction levels are abnormally high or low, or functions are either suppressed or aberrantly elevated compared to normal cell functions.
Diseases or disorders projected to respond to therapies with compounds axe in three general categories: 1) those diseases arising from either inadequate or excessive cell production, 2) those diseases arising from either inadequate or excessive cell function, and 3) those diseases resulting from either impaired or abexxant immunological screening. Immunological disorders such as autoimmune and immunodeficiency diseases (including hypogammaglobulinemia, candidiasis, acquired immune deficiency syndrome, lupus, and rheumatoid arthritis), aging (including progeria), thyroid-related disorders (including thyroiditis and hypo- and hyper-thyroidism), aystinasis, diabetes, and athero-sclerosis and related heart disease all fall within these categories. similarly, parasitic- and pathogen-induced disorders are included in the third category, at least until the cellular deficits or excesses of products or functions enabling to these organisms to escape immunological detection and elimination are determined. The above categories also include hormonal deficiency or excess, immunological deficiency or excess, infection (including parasitic, bacterial, viral, or fungal), and premature aging. The modulators as a group are efficacious, therefore, for optimizing cellular response to disease or disorder, including hormonal imbalance, immunological challenge (such as infection or infestation), and premature senescence of cells. _.
Specifically contemplated sell types for modulation, according to the invention, to ameliorate disease or disorder include: cells derived from mammalian tissues, organs and glands such as the brain, heart, lung, stomach, intestines, thyroid, adrenal, thymus, parathyroid, testes, liver, kidney, bladder, '_ spleen, pancreas, gall bladder, ovaries, uterus, prostate, and skin; reproductive cells (sperm and ova); lymph nodes, bone, cartilage, and interstitial cells; blood cells including immunocytes, cytophages such as macrophages, lymphocytes, leukocytes, erythrocytes, and platelets.
If treatment involves extraction of cells from the body, the following in vitro manipulations of extracted cells are exploitable utilities of the modulators: a) adapting to culture cells which under conventional conditions are substantially resistant to culture, i.e., those cells which have a half-life under conventional culture conditions of less than about two weeks, ox which do not express normal products or normal amounts of products in culture; b) obviating the need to fuse sells to immortalizing cells capable of long-term culture in order to obtain extended bioproduction of cell products, such as the current necessity for fusing antibody-producing splenocytes or lymphocytes to immortalizing cells for the en masse production of monoclonal antibodies; c) delaying senescence of cells and the therapeutic benefits derived therefrom, in vivo or in vitro;
d) increasing the viability of cells exposed to growth factors and/or mitogens and the therapeutic benefits derived therefrom, in vivo or in vitro; e) augmenting the biomass of cells, including stabilizing the cells) before, during, and/or after exposure to a proliferative stimulus and the therapeutic benefits derived therefrom, in vivo or in vitro; f) increasing lifsspan of cells and the therapeutic benefits derived therefrom, in vivo or in vitro; g) enhancing the bioproductivity or function of cells, or both, and the therapeutic benefits derived therefrom.
in vivo or in vitro; and h) increasing the spectrum of phenotypic expression available to cells, and the therapeutic benefits derived therefrom, in vivo or in vitro.
Specifically contemplated utility categories include a) adapting to culture cells which under conventional conditions are substantially resistant to culture, i.e., those cells which have a half-life under conventional culture conditions of less than about two weeks, or which do not express normal products or normal amounts of products in culture; b) obv9:ating the need to fuse sells to immortalizing cells capable of long-term culture in order to obtain extended bioproduction of cell products, such as the current necessity for fusing antibody-~producirig spleno~-cytes or lymphocytes to immortalizing cells for the g,~ masse production of monoclonal antibodies; c) delaying senescence of cells in culture; d) increasing the viability of cells exposed to growth factors and/or mitogens in culture; e) augmenting the biomass of cells in culture, including stabilizing the cells) before, during, and/or after exposure to a proliferative stimulus; f) increasing lifespan of cells in culture; g) enhancing the bioproductivity or function of cells in culture, or both; and h) by increasing the spectrum of phenotypic expression available to cells in culture.

d4 The lifespan of cells in culture is typically charac-terized in terms of population doubling level (PDL) of the cells, wherein each level represents a new generation of the cells. The time required for a population of cells to double is termed "generation time" (Tg), Which varies with the growth stage of a given cell type. Undex conventional culture conditions, each cell type has a lifespan characterized by a predictable number of population doubling levels, which are substantially the same fox ail healthy sells of a given type. Certain human calls, fox example, under conventional culture conditions typically double in population from about 4o to 45 times before they senesce and stop normal growth; T~ increases, and death generally occurs at about PDL 50:
In accoxdance with one aspect of the present invention, the onset of senescence is delayed in cells within the scope of the invention by exposing these cells in conventional growth medium to one or more of the vitaletheine modulators described above. By this process of the invention, the population doubling level attainable by a given cell type in culture before the onset of senescence and death increases significantly. At these high population doubling levels, the cell biomass is greatly in-creased, and the life expectancy of the cells is significantly extended; an increase from PDL 45 to PIlL 105, for example, is achievable for human cells according to this process; this represents an increa6e in total call mass as compared to biomass obtainable by conventional culture methods by a facaor of 2~°.
Further, the peak production period for cellular products is significantly prolonged, with optimization of other cellular functions. Additionally, the vitaletheine modulators of the invention are capable of eliciting enhanced aellulax~ response to chemical, biochemical, ox other stimuli, including the expression of functions different or additional, or both, to those expressed by the same type of cells at comparable stages of growth ~ vivo or under conventional culture conditions.
In order to rectangularize the life cycle of cells in culture, e.g., optimize growth and maturation of cells and minimize the stages of senescence and death, it is preferred that the cells be exposed to the vitaletheine modulators of the invention before the onset of senescence. Since cellular aging is a gradual procedure, senescence may to some degree be arrested even if the cells are exposed to modulator at a later stage in S the life of the cells, depending upon the particular cell type, culture conditions, and other factors. However, senescent cells are less viable and productive by definition, so maintaining them at this late stage of the lifespan is counterproductive for most aspects of the invention. Clearly, if the study of senescence 10 3s of primary concern then maintenance of the cells at this stage is of interest. Consequently, for optimum results in most instances, it is preferable to expose the cells to modulator as early in their life-cycle as is convenient.
Cells which are generally considered not amenable to . ' 15 culture are adapted to culture by exposure to adaptive amounts of the modulators of the invention. Cells within the scope of this embodiment of the invention include cells which have a short lifeapan under conventional culture conditions (e:g., from a few hours up to about a few weeks, for example, from about two hours 20 to two weeks), or which do not function normally in culture (e. g., wherein ~,,lr viya cell bfoproduction of hormones, enzymes, or other bioproducts is partially or substantially completely suppressed ,~ vitro). Normal cells which do not in one or more respects exhibit ~1 v~vo behavior in culture, even under optimum 2S culture conditions, as evidenced, for example by a foreshortened Iifespan or abnormal cell function, are herein referred to as ~'resistant cells°. Such resistant cells are adaptable to culture by the process of the invention by exposing the cells to be cultured to a vitaletheine modulator according to the invention, 30 ab initio, preferably by incorporating the modulator into the culture medium immediately before or soon after introduction of the cells, depending upon the particular culture medium and the stability of the particular vitaletheine modulator in that medium, By the process of the invention, cellular function of 35 resistant cells in culture is significantly improved, or substantially completely restored to normal cellular function characteristic of in vivo function, and/or cell lifespan is significantly improved or substantially completely restored to at least the cell lifespan charaoteristic of i~ v v lifespans.
Further, senescence of these cells is generally delayed in the .
presence of delaying amounts of modulators, often with a concomitant increase in, and potential diversification of, cellular function. Resistant cells within the scope of the invention include a variety of known resistant cell types, for example, lymphoid, hepatic, pancreatic, neural, thyroid, and thymus mammalian cells.
In accordance with another aspect of the invention, aberrant and intractable cells are either rehabilitated or eliminated, respectively, upon exposure in viva or in vitro to modulating concentrations of compounds, and optionally, the appropriate effector cell or celia in vitro. Cells within the scope of this embodiment of the invention include cells in which bioproduction or function is impaired. Ey the process of the invention bioproduetion is significantly improved or substan-tially completely restored to at least that characteristic of normal, in viva, bioproduction; and/or the function is improved, or substantially completely restored to at least that charac-teristic of normal, in vivo, function; and/or cell growth patterns are significantly improved or substantially completely -restored to at least those characteristic of norneal sells, in vivo; and/or the aberrant and/or intractable cells are eliminat ed. Further, in accordance with the embodiment of the invention described above, onset of disease is delayed by delaying concentrations of compounds.
Culture media irn which vitaletheime modulators of the invention are to be incorporated for modulation of cell activity of cells cultured therein do not form a part of the invention.
Exemplary useful media include all known culture media and media hereinafter developed which support maintenance and/or growth of the cells therein cultuxed. such media typically comprise at least nutrients suitable for the growth of the specific cells.to be cultured, a physiological balance of electrolytes, a physio-logieal pH, and water, as necessary to support sell growth, as well as physical culture aide such as cell supports. A variety of other known auxiliaries such as antibiotics, sera, or call growth regulators may also he included in the bassal culture media into which the modulators are to be incorporated, especially those known for enhancing cell propagation, or for augmenting cell growth and/or longevity, including cell growth factors such as peptidyl hormones specific for the cells being cultured, of the type well-known in the art. These and other auxiliaries which affect cell longevity and function in some respects are optionally included in the basal culture medium providing that io they do not completely obviate the activity of the vitaletheine modulators; in fact, selective proliferation with one or more of these factors, such as, fox example, specific peptidyl hormones, in the presence of a vitaletheine modulator to stabilize the cells being generated comprises a useful technique far selective-ly enriching the cells of interest in a gross cellular extract, for examgle, organ extracts. Compounds which inhibit metabolism of the modulators may also be included.
Conventional media into which the modulators of the invention are incorporated for the practice of the invention are ZO herein referred to as "basal culture media" . Basal culture media into which the modulators of the invention axe incorporated may be employed in conjunction with any suitable culture techniques known or hereinafter to be developed, including batch ar continuous culture, perfusion culture, or other techniques, particularly those adapted to maximize sell culture, as by the continuous repienish~nent of nutrients or other media components and continuous removal of cell waste materials.
Broadly, the modulators of the invention are suitable for modulating the activity of cells in any culture medium which 3a supports the growth of these cells and which does not signifi cantly inactivate or otherwise adversely affect the function of the modulators.
The cells nay be exposed to the. modulators of the invention in any convenient fashion. The modulators nay, for example, be incorporated into the nutrient medium, or into cell support elements. The cells may also be pre-exposed to modula-tor. In a particular embodiment of the invention, the modulators 4 8 .._ are incorporated into a support material by combining the modulators with starting materials employed to prepare the supports. Tntroduction of modulators into synthetic prepolymers for tlae production of natural or synthetic supports such as hollow fiber membranes, or pregels for the produetion of gel supports, or liquefied cellulose for the production of cellulose supports, are exemplary. The modulators may be injected in any convenient physiologically acceptable vehicle, including saline and phosphate-buffered saline, in any location which doe$ not result in the irreversible inactivation or maladsoxption of the modulator, including i.v., i.p., s.c., and i.d. Likewise, the modulators may be administered in any other fashion which does not result in the irreversible inactivation or maladsorption, such as orally with the appropriate additionally optional entero-coating, xectally, nasally including sprays, and dermally including patches.
Culture media employable with the modulators of the invention include known basal media optionally supplemented with protein components, particularly serum, e.g., fetal or new-barn calf serum. Exemplary media include Eagle's Basal Medium=
Eagie's Minimal Essential Medium; Dulbecco's Modified Eagle's Medium; Ham's Media, e.g., F10 Medium; F12 Medium; puck's N15 Medium, Puck's N16 Medium; Waymoth's MB 7521 Medium; McCoy's 5A
Medium= RPMI Media 1603, 1634, and 1640; Leibovitz's L15 Medium;
ATCC (American Type Culture Collection) CRCM 30; MCDB Media 101, 102, 103, 104; Cl4~tL Media 1066, 1415, 1066, 14:15; and Harik's or Earl s Balanced Salt Solution. The basal medium employed, as known in the art, contains nutrients essential for supporting growth of the cell under culture, commonly iaicluding essential amino acids, fatty acids, and carbohydrates. The media typically include additional essential ingredients such as vitamins, cofactors, trace elements, and salts in assimilable quantities.
other biological compounds necessary for the survival/function of the particular cells, such as hormones and antibiotics are also typically included. The media also generally include buffers, pH adjusters,. pH indicators, and the like.

Media containing the modulators of the invention are applicable to a variety of cells, especially eukaryotic cells.
The media of the invention are suitable for culturing animal cells, especially mammalian cells; plant cells; insect cells;
arachnid cells; and microorganisms such as bacteria, fungi, molds, protozoa, and rickettsia, especially antibiotic-producing cells. xhe modulators are broadly useful to pxomote viability of living cells in a broad spectrum of so-called tissue culture media adapted for the culture of such cells. Exemplary applica-tions include the culture of cloned sells, such as hybridoma cell lines; of mammalian cells for,the production of cell products, especially proteins aad peptides such as hormones, enzymes, and immunofactors; of virally-infested cells for the production of vaccines; of plant cells in, for example, xoeristem or callus culture; of epithelial cells to provide tissue for wound healing;
of xesistant cells for medical and diagnostic use; and in media adapted for the groduction and preservation of biological organs and implant tissue.
Specific cell types useful for culture in the in vi ra pros~sses of the invention accordingly include: cells derived from mammalian tissues, organs and glands such as the brain, heart, lung, stomach, intestines, thyroid, adrenal, thymus, parathyroid, testes, liver, kidney, bladder, spleen, pancreas, gall bladder, ovaries, uterus, prostate, and string reproductive cells (sperm and ova); lymph nodes, bone, cartilage, and interstitial cells; blood cells including immunocytes, cytophages such as macrophages, lymphocytes, leukocytes, erythrocytes, and .
platelets. Additional cell types include stem, leaf, pollen, and ovarian cells of plants; microorganisms and viruses ae specified above;' and cells derived from insect or aracPsnid tissues, organs, and glands.
Culture techniques useful in conjunction with the modulators of the invention include the use of solid supports, (eapeoially for anchorage-dependent cells in, for example, monolayer or suspension culture) such as glass, carbon, cellu-lose, hollow fibex membranes, suspendable particulate membranes, and solid substrate forms, such as agarose gels, wherein the compound is caged within the bead, trapped within the matrix, ar covalently attached, i.e. as a mixed disulfide. The modulators ,' are useful in primary cultures; serial cultures; subcultures;
preservation of cultures, such as Frozen ar dried cultures; arid 5 encapsulated cells; cultures also may he transferred from conventional media to media containing the modulators by known transfer techniques.
According to the practice of the invention, cells are exposed to one or more active vitaletheine modulators, ar one or 10 more active or activatable modulators, of the Formulas I through X in an amount sufficient to promote culture of these cells ~
viva or ih vitro, as measured, for example, by significant increase in cell lifespan, viability, increase in cell biomass, increase in cell bioproductivity, delay of cell sgtle9CenC~, or 15 diversification or normalization of cell function as compared to unexposed cells, or elimination of intractable cells. Modulators which delay sell senescence ar adapt resistant calls to culture are of particular interest, as are modulators which delay or obviate disease, normalize aberrant cell behavior, andjor 20 eliminate intractable cells.
Modulators useful for promoting culture of sells i~
yitro or modulating cells in viva or ~,n vit~g according to the invention comprise active- or activatable compounds of the Formulas I through X. As used herein, "active vitaletheine a5 modulators" comprise compounds of the Formulas I through X which ~ .~ modulate cells in viva or in vitro as described herein, particularly those which prowote culture of cells ,j~ yftro, especially those which directly delay senescence of cells in a given culture and/or adapt cells to culture under the conditions 30 employed, or 'Chose which directly delay or obviate disease, normalize aberrant cell behavior, andjor eliminte intractable cells. The term "activatable vitaletheine modulatorsn as used herein refers to compounds of the Formulas a through X which era not in themselves active, but are activatable to compounds which 35 similarly modulate cells in viva or in vitro as described herein, particularly those which promote culture of cells ,~ vitro, especially those which directly delay senescence and/or adapt cells to culture under the culture conditions employed, or those which di.rectiy delay or obviate disease, normalise aberrant sell behavior, andjor eliminate intractable cells primarily by rearrangement including reversible cyclization and tautomeriza- .
tion, dehydration, hydration, salt exrshange, oxidation, andJor reduction of the compounds as described herein, either i vo or before the madulatars are incorporated in a culture medium, or by appropriate adjustment of a culture medium, for example with regard tct pH, salt, partial pressure of t7a or CC~, enzyme content, exposure to W or other radiation, and temperature. The characterization of a given modulator as either "active" or "activatable" for a particular application is dependent on a variety of factors, including environment of the cell and the cell type, and selection of modulators for optimum results is made accordingly.
In practice, it is generally preferred to employ naturally~oecurring vitaletheine modulators of the Formula II and subfornaulas thereof, as the derivatives thereof of the hormula III, et.sea.. are not believed to be endogenous compounds and their metabolic pathways are at present unknown. The naturally~
occurring modulators of the Formula II are postulated to be endogenous to a broad spectrum of cells, including animal, plant, insect, arachnid, and microorganism cells, and accordingly, mast, if not all, sells derived from these organisms are expected to have well-established mechanisms for the enzymatic activation, utilization, and metabaiism of these compounds. Thus, to maximize efficacy and minimize potentially toxin or undesirable side reactions, the use of either naturally-occurring modulators of the Formula I or vitaletheine modulators activatable to the naturally-occurring modulators in the practice of the invention is recommended, especially vitalethine, vitaletheine, or vitaletheine V4 of the Formulas IId, IIe, and Ilc.
The use of modulators according to the present invention in modulating cells i~,_y~,vo or in,vitro especially by delaying or obviating disease, normalizing aberrant cell behavior, and/or eliminating intractable cells or by promoting cell culture ~ tro, especially by delaying cellular senescence and/or adapting resistant cells to culture, f.s contemplated to .
be applicable to the broad range of cells recited, owing to the postulated near-universality of precursors tc~ the compounds of the Formula II in the metabolic pathways of at least eukaryotic organisms, and the biochemical equivalence of the non-naturally occurring homologs and analogs of Formulas III through VIII.
The effect of the modulators of the invention on cellular growth patterns fs typically concentration-dependent.
Optimization of efficacy, especially ~rith respect to cell life expectancy and maximiaation of cell function (e.g,, rate of bioproduction and/or diversity or nonaalization of function] may occur within a relatively narrow concentration range of module-tor; outside this range, cell growth patterns and/or cel3 functions may tend to approach those of untreated sells. Also, the process of the invention may be, at least in some instances, reversible; that is, for example, cells retained in culture by .
exposure to the modulators of the invention ltieyo~d their ndrraal lifespan may, for example, revert to senescence soon after failure to properly replenish thg modulator.
The amount o~ modulator eliciting the desired cellular response according to the present invention is herein referred to as an "effective amount" of modulator. optimum amounts of modulator for delaying senescence, herein referred to as "senescence--delaying" amounts, are. readily determined by introducing varying amounts of modulator nto test cultures substantially before the onset of senescence, and selecting the concentration at which the lifespan of calls in culture is maximized. As previcusly noted, an amount of modulator suffi-cient to increase, for 'example, a selected cell function is often substantially equivalent to the amount of modulator reguired to effect other modulations of sell activity. Since this may not always be the case, it is useful to adjust modulator.concentra-tion against the specifically desired end result; fox exa~eple, improved rate of cell bioproduction, improved span of cellular bioproduction, improved diversity of cellula:e function, improved delay or obviation of disease, or impxoved life expectancy of cells.

The amount of modulator required to adapt resistant cells to culture is herein referred to as an ttadapting amount~~ ' of modulator. In this instance, the lifespan of resistant cells in culture is significantly improved and cell functions are normalized by at least a threshold amount of modulator. Again, optimal adaptation andjor cell function is conveniently obtained by exposing a series of test cultures to varying concentrations of modulator until the amount of modulator required to satisfac torily grow the cells in culture has been determined. Tn this to embodiment of the invention, excess amounts of modulator will not generally affect adaptation; however, if it is desired, for example, to also delay senescence in accordance with the embodiment by the invention described supra,. excess amounts of modulator tending to decrease maximum lifespan, as previously explained, should be avoided.
As a general guideline for effective concentrations of modulator for promoting cell production according to the invention, especially far promoting cell phenotypic expression, fuxsation, and viability, delaying senescence, promoting adapta-tion of cells to culture, and particularly far delaying or obviating disease, na~rmalixing aberrant cell behavior, and/or eliminating intractable cells, from about 0.01 fg to 100 ng vitaletheine modulators) per milliliter culture, and preferably from about 0.1 to 10,000 fg vitaletheine~ modulators) per milliliter culture is recommended, or for in vivo applications from about o.i fg to i,ooo ng vitaletheine modulators) per kg body weight, and preferably from about 1 fg to 10 ng vitaletheine modulator(sy per kd bvdy weight is recommended, depending particularly on the potency of the modulator and cell densities.
When combinations of the modulators are employed, total amount of modulator will usually be within these ranges. Since the effective amount at the lower concentrations of vitaletheine modulators) recited approaches one molecule of modulator per cell, it is especially important to adjust the concentration of modulator at the lower end of these ranges according to the number of cells present, i.e., the cell density, such as the density of the culture. last preferably, a basal culture medium employed is supplemented with sufficient modulator to provide a total concentration of modulators) in the medium of from about 1 to 2 fg modulator per milliliter of medium, again depending primarily upon the potency of the modulator, the type of cell, and upon cell densities. Likewise, for fn viva applications total concentration of madulator(s) is most preferably Eros l0 fg to 10o pgjkg depending upon the potency of the modulator, the type of cell, and upon cell densities. Typically, the above concentration ranges of raodulator(s) will comprise effective amounts of modulator for cultures irrespective of cell densities, but special problems of nutrient and modulator supply and waste removal exist in confluent cultures. Consequently, confluent cultures should be avoided when possible unless special provi-sions axe made for these environmental needs. Up to ten million cells per milliliter culture is a useful range of cell concentra-tion, fox confluency increases at higher cellular densities, again depending upon the size of the sells. Typical cell densities comprise from about one hundred thousan8 to ten million cells per milliliter culture, and the above described dosages axe Zo based upon such densities. Since the effective concentration of modulator has approached one molecule per cell, the concentration of modulator is varied as the concentration of cells increases or decreases.
Replenishment of the vitaletheine modulators) to regulate cell activity as desired may be advisable. Diurnal variations in enzymatic activity of modulated cultures are notable, and diurnal or 48 hour replacement is generally recommended for most cultures, typically depending upon the stability of a particular vitaletheine modulators) in the particular environment and the particular type of cell employed.
I_, viva, there is some evidence that the compounds accumulate with prolonged treatment regimens, in which case it is advisable to diminish the concentrations administered.
Based on illustrated and non-illustrated research data, it appears that cells to be cultured according to the invention may demonstrate an inherent resistance t o extra-biological amounts of vitaletheine modulator(s). This is overcome as concentrations) are increased at a dosage at which a response is first observed, herein referred to as "threshold dosage". The response augments rapidly with dose to a maximum response at a dosage herein referred to as "optimum dosage"; beyond this point, 5 the cell response typically declines with increasing dose to that observed in unexposed cells. The dosage at which basal biologi-eal activity is restored is referred to herein as "endpoint dosage". The dosage providing a response from between about the threshold dosage and the endpoint dosage is referred to herein 10 as the "effective concentration or dosage" of the modulator.
Guidelines for the development of dose-response curves for a particular application are conveniently developed as follows:
DOS, E i?,~',,,SPO~SE CURVE D LOPMENT GUIDELINES
15 pMp~"gyina Vita~,gtheine Modulators) for belaying Senescence.
Cells of the type to be cultured according to the invention are first grown in 'a modulator-free control basal culture medium according to standard practice to measure generation time. The onset of senescence is marked by a 20 significant increase in the cell generation tine, as well-understoad in the art. Samples of the same cell type at chronologically identical stages of development are then cultured in the same medium containing a modulator according to the invention in the amounts ranging for example from about o.l 25 femtograms vitaletheine modulators) per milliliter to about 1 microgram vitaletheine modulators) per milliliter culture medium, based on exemplary cell densities of about one million cells per milliliter culture; preferably, doses of the compound in logi,e increments are used to localize the effective concentra-3D tion of any particular vztaletheine modulator. The cultures are then reexamined over a range flanking the effective dosage in less than one logn~ increments to thoroughly define the affective concentration, the threshold dosage, and the endpoint dosage far that particular culture.
35 Up to a doubling of the normal lifespan and/or presenescent life of cells in culture is commonly observable according to the process of the invention, and in many instances three-fold yr more increases in iifespan are obtainable.
Further, cells cultured according to the present process exhibit differences in phenotypic expression, thought to be more characteristic of the cells, ix~ v vo, as compared to untreated cells.
o a a e' a Biological activities to be modulated according to the invention are generally evaluated in parallel in the presence and absence of vitaletheine modulators) to establish the basal biological activities under conditions identical to the evalua tion of modulated activities. Modulators are administered by routes previously described while the control or basal group receives only the vehicle for administration. F'or example, 25 groups of 5 or more animals are treated with log(10) increments from 0.1 fg to i,oao ng vitaletheine madulator(a)/kg body weight (such as by i.p. injection in physiological saline, optionally including inhibitors of the metabolism of the modulatar(sj), periodically throughout the study, such as three times per Week.
2o Samples or measuxements are taken and preserved as the regimen is continued for at least two weeks and preferably for 15 weeks.
After compilation of the data, the response is evaluated graphically with a three dimensional surface in which the X, Y, and 2 axes are dose, week, and response, respectively. The 25 optimum concentration of modulator is easily identified fn this manner; and if inhibitors) of metabolism have been included, the study is repeated holding this concentration constant and varying the concentration of the inhibitors) to optimize the inhibi-tor s) concentrations) as well. Repeating the analysis With 30 more closely spaced ncrements of modulator and with a constant optimal dose of inhibitor, then, localizes the eptimum and effective range of concentrations for compounds.
Therapeutic effects are obtainable with as little as ido ng of inhibitor/kg body weight and with less thaw 100 pg 35 vitaletheine modulator/ kg body weight.

III. Utilitx of the Compounds lcontinued~:
8. The compounds are further useful, inte,I alia, for promoting phenotypic expression of normal and neoplastic calls, normalizing neoplastic cells, and/or eliminating these cells from the body, including, for example, reestablishing normal growth cycles, lifespans, and functions of tumor cells and immune Cells in particular, and especially of NK (natural killer] cells.
Specifically contemplated utility categories include aj enhancing the capacity of NK cells to destroy tumor cells, bj rendering tumor cells vulnerable to calls of the immune system, c) prolonging the lifespan of immunocytes belligerent to neoplastic cells, in-.v~,vo, and d) interrupting the underlying mechanisms of cell transformation from neoplastic to malignant cells.
Modulator or modulators useful for treating neoplasia according to the method of the invention camprise active- or activatable compounds of the Formulas I through X. As used herein, "active vitaletheine modulators" comprise compounds of the Formulas I through X which oar ease are effective in vivo or 30 in vitro for the treatment of neoplasia. The term "activatable vitaletheine modulators" as used herein refers to compounds of the Formulas I through X which are not in their initial form active, but are activatable by biological or other means to compounds ~rhich similarly are effective for the treatment of neoplasia ~~n vitro or ;1~_vivo, primarily by rearrangement including reversible cyclization and tautomerization, dehydra-tion, hydration, salt exchange, oxidation, and/or reduction of the compounds as described herein, before the modulators are incarporat~d in the culture medium, before the compound is administered in vivo, or by appropriate adjustment of the v tro or in vi.vo conditions, for example :with regard to pH, salt, partial pressure of oa or COz, enzyme content, exposure to W ar other radiation, and temperature.. The characterization of a given modulator as either "active" or "activatable" for a particular application is dependent on a variety of factors, including environment of the cell and cell type, and selection of modulators for optimum results is made accordingly.

In practice, it is generally preferred to employ naturally-occurring vitaletheine modulators of the Formula II and subformulas thereof, as the derivatives thereof of the Formula III e°_t.sea. are not believed to be endogenous compounds and their metabolic pathways are at present unknown. The naturally-occurring modulators of the Formula II are postulated to be endogenous to a broad spectrum of cells, including animal, plant, insect, arachnid, and microorganism cells, and accordingly, most, if hot all, cells derived from these organisms are expected to have well-established mechanisms for the enzymatic activation, utilization, and metabolism of these compounds. Thus, to maximize efficacy and minimize potentially toxic, undesirable, or even hazardous side reactions, the use of either naturally-occurring modulators of the Formula I or vitaletheine modulators activatable to the naturally-occurring modulators in the practice wr of the invention is recommended, especially vitalethine, vitaletheine, or vitaletheine V4 of the Formulas IId, ITe, and IX.
The use of modulators in vivo or in vitro aCCOrding to 2o the present invention in treating neoplasia is contemplated to be applicable to a broad range of cells, owing to flee postulated near-universality of precursors to the compounds of the Formula II in the metabolic pathways of at least eukaryotic organisms, especially humans, and the biochemical equivalence of the non-naturally occurring homalogs and analogs of Formulas III
through VIII.
The effectiveness of the modulators on neaplasia is typically concentration-dependent. Optimization of efficacy may occur within a relatively narrow effective concentration range of modulator; outside this range, neoplasia may be unaffected or exacerbated. Also, the process of the invention may be, at least in some instances, reversible; that is, neoplasia may return to untreated growth after treatment is discontinued.
The amount of modulator eliciting the desired biologi cal response according to the present invention is herein referred to as an "effective amount" of modulator. Optimum amounts of modulator fnr the treatment of neoplasia are readily ' 59 i7 deter~onined by introducing varying amounts of modulator into test cultures or, vivo, and selecting the eoncentrat~i.on at which ' tumors are inhibited.
The modulators may be administered directly to the organism, for example, the mammal, according to the process of the invention, in amounts sufficient tn promote the desired biological response by conventional routes, such as parenterally, in any location which does not result in the irreversible , inactivation or maladsorption of the modulator, including i.v., to i.p., s.c., and i.d. hikewise, the modulators may be adminis-tered in any other fashion which does not result in the irrevers- ' .
ible inactivation_or maladsorption, such as orally with the apprapriata~ additionally optional entero-coating, rectally, nasally including sprays, and dermally including patches.
standard carriers not affecting compound i-ntec~rity are useful for administration of vitaletheine modulators, such as physiological saline.
The modulator is administered indirectly according to the process of the invention by removing iramunocytes from the 2o afflicted body, treating them in culture as described herein, and reinjecting them according to standard procedures as described in, for example, Immure ResnQnses to Metastases, volume II, chapter ii, 198, cRC press, Inc., Boca Raton, Florida, vsA, incorporated herein by reference. Preferably the cytotoxicity Z5 of the immunocytes towards tumor cells is further enhanced by additional in vitro exposure of the cells either to the tumor cells, especially those derived from the afflicted mammal, or to inhibitors of the metabolism of the modulator or modulatorr>, or to a combination thereof, prior to reinjection. Enhancement of 30 cytotoxicity of immunocytes towards tumor cells is described for example, supra.
According to the method of the invention, cells may be exposed to the modulator or modulators, in vitro, in any convenient fashion. The modulators may, for example, be 35 incorporated into the nutrient medium, or into cell support elements. The cells may also be pre-exposed to modulator. In a particular embodiment of the invention, the modulators are incorporated into a support material by combining the modulators with starting materials employed to prepare the supports.
Introduction of modulators into synthetic prepolymers for the production of natural or synthetic supports such as hollow fiber 5 membranes, or pregels for the production of gel supports, or liquefied cellulose for the production of cellulose supports, are exemplary.
Culture media in which vitaletheine modulators are to be incorporated for modulation of cell activity of cells cultured 10 therein do not form a part of the imrention. Exemplary useful media include all known culture media and media hereinafter developed which support maintenance andJor growth of the cells therein cultured. Such media typically comprise at least ' nutrients suitable for the growth of the specific ceps .to be 15 cultured, a physiological balance of electrolytes, a physiologi-cal pH. and water, as necessary to support cell growth, as well as physical culture aids such as cell supports. A variety of other known auxiliaries such as antibiotics, sera, or cell growth ' regulators may also be included in the basal culture media into 20 which the modulators are to be incorporated, especially those known for Enhancing cell propagation, or for augmenting cell growth and/or longevity, 'ancluding cell growth factors such as peptidyl hormones specific for the cells being cultured, of the type well-known in the art. Thesa and other auxiliaries which 25 affect cell longevity and function in some respects are optional-ly included in the basal vulture medium providing that they do not completely obviate the activity of the vitaletheine modula-tors; in fact, selective proliferation with one or more of these factors, such as, for example, specific peptidyl hormones, in the 3t7 presence of a vitaletheine modulator to stabilize the cells being generat~l comprises a useful technique for selectively enriching the cells of interest in a gross cellular extract, for example, organ extracts. Compounds which inhibit metabolism -of the modulators may also be included.
35 conventional media into which the modulators are incorporated for the practice of the invention are herein referred to as "basal culture media": Basal culture media into which the modulators are incorporated may be emgloyed in conjunction with any suitable culture techniques known or hereinafter to be developed, including batch or continuous culture, perfusion culture, or other techniques, particularly those adapted to maximize cell culture, as by the continuous replenishment of nutrients or other media components and continuous removal of cell waste materials.
Broadly, the modulators are suitable gor modulating the activity of cells in any culture medium which supports the grpwth of these cells and which does not significantly inactivate or otherwise adversely affect the function of the modulators.
Culture media employable with the modulators include known basal media optionally supplemented wit3i protein components, particu-larly serum, e. g. , fetal Or new-born calf serum. l3xemplary media include Eagle's Hasal Medium; Eagle's Minimal Essential Medium;
Dulbecco's Modified Eagle's Medium; Ham's Media, e.g., F10 Medium; F12 Medium; Fuck's N15 Medium, Puck's N16 Medium;
Waymoth°s MS ?521 Medium; McCoy's 5A Medium; RPMI Media 1603, 1634, and 1640; Ireibovitz's L15 Medium; ATCC (American Type Culture Collection) CRCM 30; MCDH Media 101, 102, 103, 104; C1~RL
Media 1066, 1415, 1066, 1415; and Hank'e~ or Earl's Balanced salt Solution. the basal medium employed, as known in the art, contains nutrients essential for supporting growth of the oell under culture, commonly including essential amino acids, fatty acids, and carbohydrates. The media typically include additional essential ingredients such as vitamins, cofactors, trace elements, and salts in assimilable quantities. Other biological compounds necessary for the survival/function of the particular cells, such as hormones and antibiotics are also typically included. fhe media also generally include buffers, pH adjust-ers, pH indicators, and the like.
Media containing the modulators are applicable to a variety of cells, especially eukaryotic cells. The media are suitable for culturing animal cells, specifically mammalian cells and especially human cells. Specific cell types useful for culture in the processes of the invention accordingly include:
sells derived from mammalian tissues, organs and glands such as ' 62 the brain, heart, lung, stomach, intestines, thyroid, adrenal, thymus, parathyroid, testes, liver, kidney, bladder, spleen, pancreas, gall bladder, ovaries, uterus, prostate, and skin;
reproductive cells (sperm and ova} ; lymph nodes, bone, cartilage, and interstitial cells; blood cells including immunocytes, cytophages such as macrophages, lymphocytes, leukocytes, erythrocytes, and platelets.
Culture techniques useful in conjunction with the modulators include the use of solid supports, (especially for anchorage-dependent cells in, for example, monolayer or suspen sion culture) such as glass, carbon, cellulose, hollow fiber membranes, suspendable particulate membranes, and solid substrate farms, such as agarose gels, wherein the compound is caged within the bead, trapped within the matrix, or covalently attached, i.e.
as a mixed disulfide. The modulators are useful in primary cultures; serial cultures; subcultures; preservation of cultures, such as frozen or dried cultures; and encapsulated cells;
cultures also aay be transferred from conventional media to media containing the modulators by known transfer techniques.
ZO As a general guideline for effective concentrations of modulator fox treating neoplasia, from about 0.01 fg to 100 ng vitaletheine modulator{s} per milliliter culture, and preferably from about 0.1 to 10,000 fg vitaletheine modulators) per milliliter culture is recommended, or for ,~, vivo applications from about o.i fg to 1,000 ng vitaletheine modulator{s) per kg body Weight, and preferably from about 1 fg to 10 rig vitaletheine modulator{s) per kg body weight is recoansended, depending particularly an the potency of the modulator and cell densities.
When combinations of the modulators are employed, total amount ' of modulator will usually be within these ranges. Since the effective amount at the lower concentrations of vitaletheine modulators) recited approaches one molecule of modulator per cell, it fs especially important to adjust tins concentration of modulator at the lower end of these ranges according to the number of cells present in culture or in vivo, i.e., the target cell density, such as the density of leukemia cells, as readily determined by standard methods. Most preferably, the basal culture medium employed is supplemented with sufficient modulator to provide a total concentration of modulators) in the medium of iron about 1 to 2 fg modulator per milliliter of medium, again depending primaxily upon the potency of the modulator, the type of cell, and upon target cell densities. Likewise, for in vivo applications total concentration of modul~ator(s) is most preferably from io fg to 10o pg/kg depending upon the potency of the modulator, the type of cell, and upon target cell densities.
2'ypically, the above concentration ranges of modulators) will comprise effective amounts of modulator for cultures irrespective of cell densities, but special problems of nutrient and modulator supply and waste removal exist in confluent cultures. Conse-quently, conflu~nt cultures should be avoided when possfble unless special provisions are made for these environmental needs.
Bp to ten million cells per milliliter culture is a useful range of cell concentration, for confluency increases at higher cellular densities, again depending upon the size of the cells.
Typical cell densities comprise from about one hundred thousand to ten million cells per milliliter culture, and the above described dosages axe based upon such densities. Since the effective concentration of modulator has approached one molecule per cell, the concentration of modulator is varied as the concentration of cells increases or decreases.
Replenishment of the vitaletheine modulators) to regulate biological activity as desired may be advisable.
Diurnal variations in enzymatic activity are ,notable, and diurnal or 4s hour replacement is generally recommended, typically depending upon the stability of a particular vitaletheine modulators) in the particular environment and the particular .
3o type of cell targeted.
The method of the invention is useful for reducing ~
v vo both solid (non-hematolymphaid) and soft (hematolymphoid) tumor burden, particularly in mammals, and inhibiting intra-vascularization of tumor cells, especially cells of metastasizing .
tumors. The compounds are thus broadly useful for reducing tumor burden, by inhibiting tumor growth or by inhibiting tumor metastasis, or both. In particular, they are contemplated to be effective either alone or in combination with a-alethine or other metabolites or inhibitars of their metabolism, against a broad spectrum of malignant tumors, especially tumors such as melano-mas; myelomas; lymphomas; ieukemias; and carcinomas; including ovarian tumors; cervical tumors; uterine tumors; breast tumors; .
lung tumors (small cell and non-cell carcinomas); colon and stomach tumors; hepatocelllular tumors; pancreas, midget, liver, bone, bladder, and prostate tumors; brain tumors (primary and secondary); larynx and oral cavity tumors; skin tumors; and Hodgkin~s disease. The modulators are contemplated as useful inter alia in the treatment of neoplasia 1)prophylactically; 2j as a primary therapy for inhibiting tumor growth, particularly that of slowly-growing tumors; and 3) as a supplemental therapy pursuant to surgical intervention for removal or debulk$ng of tumors, particularly virulent or primary tumors. Treatment with the.modulators has been found to inhibit development of aggres-sive tumors, diminish tumor mass, regress tumors, and inhibit tumor metastasis. It is recommended that anti-tumor therapy commence at the earliest tumor stage possible, particularly to avoid peripheral physiological complications caused by the , presence or metastasis of large tumors, and to diminish the systemic burden of tumor debri subsequent to the implementation of an effective regimen.
Based on illustrated and non-illustrated research data, it appears that neoplasia to be treated according to the invention may demonstrate an inherent resistance to extra biological amounts of vitaletheine modulator(s), in vitro or in V1V0. This is overcome as cancentration(s) are increased at a dosage at which a response is first observed, herein referred to as ',threshold dosage°°. The response augments rapidly with dose to a maximum response at a dosage herein referred to as "optimum dosage°°; beyond this point, the therapeutic response typically declines with increasing dose to that observed prior to the exposure. The dosage at which basal biological activity is .
restored is referred to herein as "endpoint dosage°~. The dosage providing a response from between about the threshold dosage and the endpoint dosage is referred to herein as the "effective concentration or dosage" of the modulator. For example, polymerization of the vitaletheine, in vivo or fn vitro, to vitaletheine Vd at dosages above the optimum dosage may result in a decline in the desired response, and may additionally cause 5 proliferation at concentrations greater than the endpoint dosage.
Guidelines for the development of dose-response curves for a particular application are conveniently developed as follows:
DOSE RESPONSE CURVE DE~jELOPMENT GUIDELINES
10 Emnloyinq Vitaletheine Modulators) ua in vitro neotolasia applications.
Targeted cells according to the invention are first grown in a modulator-free control or basal culture medium according to standard practice to measure tumor cytotoxicity.
15 Samples of the same cell type at chronologically identical stages of development are then cultured in the same medium according to the invention containing a modulator in the amounts ranging for example from about o.ol femtograms vitaletheine modulators) per milliliter to about 1 microgram vitaletheine modulators) per 20 milliliter culture medium, based on exemplary cell densities of about one million cells per~milliliter culture; preferably, doses .
of the compound in log~~~ increments are used to localize the effective concentration of any particular vitaletheine modulator.
The cultures are then reexamined over a range flanking the 25 effective dosage in less than one log~~~ increments to thoroughly define the effective concentration, the threshold dosage, and the endpoint dosage for that particular culture. once the i~r vitro treatment is optimized, the cells are reinjected to inhibit or .
regress tumor as determined by standard methods such as palpa°~
30 tion, enzyme or specific protein assay, or magnetic resonance or other imaging procedures.
Fmp~,ovinu Vitaletheine Modulatorlsl in iri v ~o neaplasia ~,p lications.
Preferably, the biological activity of the modulator 35 to be employed is evaluated by standard procedures using a control group to establish the basal biological activities under conditions identical to the evaluation of modulated activities.
Modulators are administered by routes previously described while the control ar basal group receives only the vehicle for administration. For example, groups of 5 or more animals are treated with log~,~ increments of from 0.01 fg to 1,000 ng vitaletheine modulator(sj/kg body weight (such as by i.p.
infection in saline, optionally including inhibitors of the metabolism of the modulator(s)), periodically throughout the study, such as three times per week. Tumor samples or measure-ments are obtained and preserved as the regimen is continued for at least about two weeks and preferably for about 15 weeks or mare. After compilation of the data, the response is evaluated graphically with a three-dimensional surface in which the X, Y, and Z axes are dose, week, and response, respectively. The optimum concentration of modulator is easily identified in this .
manner as a depression an the surface when 2 is tumor develop went. When an inhibitor(sj of metabolism has been included, the optimum dosage of the compound is determined, then the study is repeated holding this concentration of the compound constant and .
varying the concentration of the inhibitor(sj to optimize the inhibitors) concentration(sj as well. Repeating the analysis with more closely spaced increments of modulator, for example half logtlg, and with a constant optimal dose of inhibitor, thenB
localizes the optimum and effective range of concentrations for the compounds. Therapeutic effects are expected with as little as 100 ng of inhibitor/kg body weight and with less than 100 pg -vitaletheine modulator/kg body weight.

BXAMPLES
EXAMPLE x . Bynthes j,~",of N,,I~ ~ -bis- f CB21-8-~~etln~ee iS,S~-Gist(N-carbobanEOXy~-alanyll-2-amir~aethyll Disulfi e?
A solution of dicyclohexyloarbadiimide (23.3g) was added to a solution of N-CBZ-S-alanine (24.84g) and N-hydroicy-succinimide (12.92g) in a total volume of about 500 ml of dry 10% acetonitrile in dichloromethane. Dicyclohexylurea (2A.51-g) precipitated as a by-product upon formation of the active ester. The active ester was dried to an oil and triturated with anhydrous ethyl ether. The precipitate was resuspended in dlchloromethane and additional dicyclohexylurea was allowed -to precipitate. The resulting dichloramethane solution of active ester was filtered and added to a previously prepared --solution of cystamine (8.5g). The desired product, N,N~-bis-(CBZ)-B-alethine precipitated from this mixture. The mother , liquor, anhydrous ether and dichloromethane extracts of the product, and the anhydrous ether extract of the active ester, above, were dried and recombined to augment the yield of '' product. N,N'-bis-(CBZ)-B-alethine was practically insoluble in water, hot ethyl acetate, and hot ether, and these were used to further extract impurities. The product was recrys-talli$ed from dimethyl sulfoxide with acetonitrile (or water), and again rinsed with ethyl acetate and ether. This last 2~5 process resulted in a 1°C increase in melting point to 180-181°C (uncorrected). Yields of N,Na-bis-(CBZ)-8-alethine of 8S-90% were routinely obtained, and near-quantitative yields ' are possible. When dried over PROs, .~11 vacuo, the product appeared to retain one mole equivalent of water, and was analysed accordingly as the monohydrate, Anal. Ca , for c~H~N,ObSz.Hic?: C, 53.78; H, 6.25; N, 9.65.
Found: C, 5A.23; H, 6.56; N, 9.66. sample analyzed by Ruby Ju, Department of Chemistry, University of New Mexico, Albu-querque, New Mexico.

Example II. ~nthesis an~Char~~ct~rization oi° the Benzvl Deri_vatiye of V,f~,~ett~g A. Synthg~~is. The (allowing reagents were added with mixing in the order listed to an Erlenmeyer flask (500 ml}: N,N'-bis-(aarbobenzoxy}-B-alethine (0.76g) from Example I, above, dimethyl sulfoxide (0.75 ml}, N,N'-dirrtethylformamide (0.75 m1}, pyridine (1 ml), chloroform (21 ml), water (150 ml}, and iodine (3.3 g). Upan addition of the iodine the pH
began to decrease, and was maintained at 5.7 by slowly adding zinc oxide (0.3 to 0.4 g). It was desirable to maintain this slightly acidic pti to optimize reaction rates. This mixture allowed controlled reaction, continuous extractian of the intermediate product from the organic reagent phase into the aqueous phase, and continuous monitoring of the pFI of the aqueous phase. When the reaction began to subside, which was indicated by a stabilization of pH, the aqueous phase was removed and subjected to repeated extractions with chloroform until no color was evident in the organic phase. Periodically during these extractions, the pH was readjusted to 6.0 with a minimum amount of Zno. When completely extracted and neutral-ized to pH 6.0, the aqueous phase was dried on a rotoevapor-ator at low temperature (<40°C), to a viscous oil. During this process, the organic phase of the reaction mixture was re-extxacted with water to recover residual intermediate product, which was subsequently extracted with chloroform, neutralized with Zno, and dried with the first aqueous extract.
This stage in the synthesis represents a branch point for the synthesis of the desired compound; at this point, either the desired compound or the benzyl derivative thereof can be obtained. For example, either vitaletheine V4 (Example III and formula IX} or the benzyl derivative of vitaletheine of the Formula VIII can be produced at this stage.
To obtain the benzyl derivative of vitaletheine, the aqueous extracts obtained as above were treated with ten volumes of acetonitrile to precipitate the benzyl derivative as the primary product.

B. Characterizat~gn of the ~p~yl Deriyative Qf Vitaletheine. The benzyl derivative obtained above had ap-proximately the same molecular weight as the blocked alethine starting matexial. However the derivative was unlike N,N~-bis-(CBZ)-R-alethine in many respects: it was soluble in water; it had unique (}3CJ- and [1N)-NMR spectra; and its IR
spectrum was likewise distinct. The benzyl derivative was purified as the calcium salt, but this difference from the zine.salt of vitaletheine V$ (below) could not account for the extremely high melting point of the former; the benzyl deriva tive melted at temperatures in excess of 300°C, while the starting material melted at 180-181°C (uncorrected). The NMR
spectra of the zinc and calcium salts of the benzyl derivative were quite similar, evidence that salts alone could not ac-ts count for these differences.
The spectra of the benzyl derivative were not con-sistent with thiazolidine or cyclic-urethane structures, and no detectable disulfide or thiol was present, suggesting that like vitaletheine V,~, the ben2yl derivative was formed by the nucleophilic attack involving sulfur on one of the carbonyl carbons in each monomer. unlike vitaletheine V4, the predomi-nant polymer in the product benzyl derivative was identified as a dimer, probably farmed by attacks of each monomer on the carbonyl carbon of the other, as described above. The quater-nary carbons present appeared identical, and were riot shifted upfield (**) in the NMR spectrum, in contrast to the pro-nounced upfield shift of the quaternary carbon atoms present in the vitaletheine tetramer, indicating fewer structural constraints in the benzyl derivative dimer than in the vitale-theine tetramer. Elemental analysis indicated additional material had crystallized with the benzyl derivative, and good correlation was found for inclusion in the dimer of 2 mole equivalents of calcium ion and one mole equivalent of oxygen per mole of the dimer. This was consistent with the presence of a calcium oxide bridge between two dimers, stabilised by hydrogen bonding. The following was the result of elemental analysis for the benzyl derivative obtained above, correcting for the presence of the calculated oxygen and calcium ion:
syca. for C26Ii~N40gSz, 2 Cat+.O-: C, 45.20; H, 4.95; N, 8 . 1 1 .
5 Fo t C, 44.97; H, 4.98; N, 8.04. Sample analyzed by Ruby Ju, Department of Chemistry, 'Cniversity of New Mexico, Albu-querque, New Mexico.
Example III. S es' arac a i at o V t et e' V .
A. ~yrnthesis. The benzyl group was removed by 10 repeatedly exposing the dried aqueous extracts obtained in T~xample ZIA to ultraviolet light (Pen-ray quartz lamp, Ultra Violet Products, Ins., Cambridge, U.K.) and extracting with chloroform until no color developed under UV irradiation, and no color was extractable into chloroform. UV irradiation is 15 particularly recommended for effectively obtaining product substantially devoid of aromatic moieties, without causing more serious and inactivating rearrangements and decomposi-tiona, as described previously. The product (when completely free of aromatics) was dried, neutralized in water with Zn4, 20 and recrystallized from dimethylsulfoxide with aeetonitrile to yield the zinc salt of vitaletheine Vi.
H, Chyracterizat3on of Vitaletheine Ve . Vitale--theine V4 was likewise distinct with reference to both the starting material and the benzyl derivative. Obtained in 25 greater than 50% yield from the above procedure, it melted with decomposition at 233-235°C (uncorrected). Evolution of gas signified decomposition of the molecule; the evolved gas (COz) was trapped by bubbling through a saturated solution of Ba{OH)z under Nz, with recovery of BaC09. Decomposition of the 30 molecule on heating was consistent with the presumptive ther-mal lability of the postulated carboxyamino structure, as was the evolution of CQZ upon heating, and the recovery of the trapped COz as the insoluble barium carbonate. The possibility that the evolved gas resulted from decomposition of zinc ' carbonate contaminating the vitaletheine V,~ was deemed unlike-ly, since this salt decomposes with COz evolution at 300°C.
The spectral evidence likewise indicated a structure unique to vitaletheine V~, comprising covalent attachment of the carbon in question (2) to the B-aletheine moiety. Concomitant with the evolution of COz, losses of a sharp H-H stretch resonance at 3290 amv and other resonances associated with the carboxy-amino structure were observed in the IR spectra.
20 Vitaletheine V4 as prepared was somewhat hygroscop-ic, possibly exacerbated by residual dimethylsulfoxide. The following elemental analysis reflected the propensity of the molecule to gain water:
Jynal. calcd. for C~H~N80tzSa.2 Zn++.8 gyp: C, 27.72; H, 5.82; N, 18.98, oun : C, 28.56; H, 5.94; N, 10.96. Sample analyzed by Ruby Ju, Department of chemistry, University of New Mexico, Albuquerque, New Mexico.
The results of several different analyses indicated that the vitaletheine dimer contained 1 Zn*z, the trimer con-tamed 1.5 :n*z, and the tetramer contained 2 Zn*Z per mole of polymer.
Example IV. Synthesis and c~aractgrization of Vitalp,$~j,~e via fi-a 1 ethine .
A. Synthesi,~ o~B-alethine.2~iC7~ or N,N'-bis-(B-alanyl)-cystaraine or N,N'--bis-(f3-alanyl-2-aminoethylj disul-fide. Complete removal of the carbobenzoxy croup was acco~a-plished according to procedures described in J.Am.Chem.Soo.
86: 1202-1206 (1964}, incorporated herein by reference. After deblocking with four equivalents of hydrogen bromide in gla-cial acetic acid per mole of the N,N~-bis-(CHZ)-B-alethine (from Example I, above) for 15 hours, the B -alethine was purified by precipitating with acetonitrile, rinsing with anhydrous ethyl ether, resuspension in water and filtering, and precipitating the mixed salts with acetonitrile. Initial yields were in excess of 80% theoretical. The B-alethine was converted to the hydrochloride salt by passing the preparation over a 30 ml X 15 cm long column of Dower AG 1X8 (chloride form) (Dow Chemical Corg., Midland, MI) which had been previ-ously prepared by eluting with 1 M potassium chloride and rinsing thoroughly with DI (de3oni2ed) water. Neutralization with Ca(OH)Z and recrystallization of the 8-alethine hydrochlo-ride from water with acetonitrile resulted in fine needles which melted at 224-225°C (uncorrected).
t0 ~: Ca cd, for ClotinN40zSz.2HCl: C, 32.69; FI, 6.59; N, 15.25.
Found: C, 32.52; H, 5.69; N, 15.32. Sample analyzed by Ruby Jn, Department of Chemistry, University of New Mexico, Albu-querque, New Mexico.
B. Syntj~esi~ of Vita:~ethine. To a suspension of Zn0 (6.5 mg from King's Specialty Company, Fort Wayne, Indi-ana, U.S.A.) and fi -alethine (6.35 mg tram Example IV. A.
shove) in pyridine (12.g mg from Fisher Scientific, Fair Lawn, N.J., U.S.A.) and dimethylsulfoxide (0.5 ml from Sir~na Chemi-cal Company, St. Louis, M.O., U.S.A.), and in a vessel equipped with a gas trap containing sodium hydroxide (at least lid), was added 0.2 ml of a solution of phosgene (20% in tolu-en~ from Fluka Chemical Corp, Ronkonkoma, N.Y., USA). Packing of the reaction vessel in dry ice controls the exothermic reaction and improves yields of large-scale preparations.
After 48 hours of reaction the excess phosgene was blown into the alkali trap with Nx. The product was precipitated in the vessel with acetonitrile (approximately.50 ml,s from Fisher Scientific, Faix Lawn, N.J., U.S.A.). vitalethine can be recrystallized from water with acetonitrile.
C. Characterization of Vitaiethine. Unlike the starting material, B-alethine which melted at 224-225°C (uncor-rected), the vitalethine powder sintered and turned brown at 215-220°C, but did not melt until 242°C (uncorrected) at which point obvious decomposition and evolution of gas occurred.

This behavior resembled that of vitaletheine V" iri that gas was also evolved upon melting of the polymer. The infrared spectrum of the two compounds ware likewise similar, but the vitalethine spectrum did not exhibit the C-o stretch bands observed in the polymer. Both compounds lost infrared reso~
nances associated with the carboxy-°amino group upon thermally labilizing this moiety, This was particularly true of vital-ethine, fvr major peaks disappeared at 1600 and 1455 CM'1 (xesonances for the ionized carboxylic moietlr), and losses in IO the fine structure in the xegions 2800 to 3300 CM'i and 900 to 1360 Cl~t~ (i.e., those associated with the N-Hand C-N moieties of the carboxy-amino group) were also apparent upon heating at 242°C.

F.xAMPLE V

~t3C~'.~*L.iR7 s =SEC G1F y.~,,~~~F!L')iINE. Vs ' W ~~

~1D RELA'f,~~COMPOUNDS

"

~( 3C1..~ . . .

s b c d a f 8-CHi CHI-NIi-N~C~0 b=C-CtgCtiZ N Ft-N-Ca0 \o-A-alethine37.59 39.04172.79 32.9 36.71 VitalAtheine V~ 36.66 95.9347.06*44.?5 32.96 172.73 50.39 39.41*38.51 Benzyl , derivative33.99 95.76156.46* 48.36 34.67 172.25 r'~ i ..~$ .

a b c d a f S-CH= Qi~-NIi-N-C~ ~C-CHa~t"'N H-N-~

..

b-alethine*2.524 3.0942.694 3.367 8-aletheine2.512 3.0842.695 3,3?2 (tn+a) ..

A-aletheine2.512 3.0872.687 3.366 .

(+Is) Vitaletheine ' V4 tD~O) 2.502 3.0812.937 3.415 (DMSO-D~)2.200 2.7637.84 2.418 3.131 7.38 Benzyl derivative to~o~ z.z3z 3.ao1z.a41 3.330 (pMSO-D6)2.210 3.1967.84 2.593 3.309 7.247 bio-(CBZ)-9-alethine (DMA-D6) 2.740 3.309$.085 2.254 3.192 7.24 Rednvtaae factor 2.71 3.08 2.90 3.28 ~=na~tx~e> , ~

. S-CHz CH1-NH-N~C=O OuC-Cti~CFIy-H 8-N-, a b c d e. f *a-alethine was reduced with REDUCTACRYL~*
(a propri-etary reducing agent available front Calbiochem, San biego, CA, USAj in Zno to form heine. The latter the presence B-alet of reacted provide With a IZ to third reference compound, probably the sulfenyl iodide, IR 1 em'' ) a b c d a ! -S-CH= CHs-PI H-N-CEO ~C-CH= CH: N
H N~0 Cr 5 Vitalethine 3170w 3290m 1550w 1560a 1&DOm 1455a Yitalartheina IO V4 7i0w 3080a 3290a .

1530m 1560a 1253m 1650e 15 Hsnzyl 692-570w 3308e 3308e derivative 1542e 1542s 1635e 1253m bla-(C88)- 3345e 3345a $0 B~alalhina 1545m 1535e 16408 1290m 1682et B-alethine 660w 3250w 3210v 1555w-a 29TOe-w 25 1286m 1452e 1620s 1620s 1128e a b c d a f S-CH, CFIz-P1 H-M-Cs0 OC-CHs CHZ-N -N-H

30 Vitaletheine V,, and vitalethine were unique in that resonances associated with the moiety "f"~abov~e disappeared when the compvunda melted and decomposed at 233--235C (uncarrected~

and 242C, respectively, presumably loss of COl. Yn due to vitaletheine V4, these losses oocurredconcomitant losses without 35 in the regions designating a (-C-0-)ythus the decarbox-polymer;

ylated form of Vitaletheine V, appearedbe an oligomer o~
to B-aletheine similar to the undecarbaxylated polymer, but lacking the carboxy moieties.
Peaks for Vitalethine: 3290ra, 317ow with shoulder at 3 100, 2990m, 1664x, 1600w, 1565m, 1455x, l4lOw with 1400 shoulder, 5 1330w with 1310 shoulder, 1260m with 1230 shoulder, 1190w, 1135m, I100m with 1090 shoulder, 103om-s, 955m.
Peaks for heated Vitalethine: 3120s (broad , 1655x, 1550m, 1405s with shoulders at 1450 and 1.390.
The IR spectrum of vitaletheine V4, following, Was 10 shiFted by exchanging seetonitrile for water of hydration in the complex.
Peaks for Vitaletheine V,: 3290s, 3080sibroad to 2500, 1650s, 15608, 153om, 1453w, 1417w, 1393w, 1346w, 1318w, 1253m, 1 190s, 1170x, 1115w/shoulder, 1040x, 1030x, 956m, 790m with shoo lder, 15 709wJbroad, 612m/sharp, 526m. These shifts approximated those observed in the spectrum of R-alethine upon neutrali$ation, below.
B-alethine was unusual in that changes in pH, i.e., neutralization with Ca(OH)a, caused pronounced shifts iia the 20 positions and intensities of bands.
Peaks (HCl salt): 32705, 3170x, 2970s, 2700w, 2550w, 2 020w, 1557x, 1595m, 1560s, 1450x, 1409m, 1390w, 1354w, 1325m, 1300w, shoulder/1252m/shoulder, 1188m, 1129m, 1097m, 1079w, 1030w, 950w, 905w, 829m.
25 Peaks (nautraiized): 3250w, 3180w, 2940m/broad, 2375x, 2 230x, 2157s, 1936w, 1620s, 1555w, 1462x, 1432 shoulder, 1400m, 1342m, 1286m, 1217m, 1188m, 1128x, 1020m, 810w, 719m, 660w.
The benzyl derivative displayed considerable homology with vitaletheine v,.
30 Peaks: 3308s, 3060w, 2942w, 1684x, 1635s, 1542x, 1447m, 1380w, 1335w, 1286w, 1253m, 1193x, 1170 shoulder, 1080m, 304Om, 980w, 738m, 692m, 609m, 550w.

Bis-(CB2j-B-alethine displayed little of the C-0 resonances around 1200 observed in vitaletheine V4 and the benzyl derivative. Peaks: 3345s, 3310s, 1682s, 1640x, 1545m shoulder, 15355, 1450w, 1427w, 1375w, 1332m, 1270m, 1231m, 1178w, 1120w, 1030m/broad.
In the following Examples, all cells were cultured at about 37°C for the specified time.
EXAMPLE iFI: Adaptation of Human Natural ~i.'Llar ,(NKj, Cep to Culture Human NK cells were purified as described in 3.Exp. I~,d.
~ 96 ,: 99-113, 1989. A standard culture medium for the cells was prepared, comprising RPMI 1640 (Rosewell Park Memorial Institute, from Whittaker M. A. Bioproducts, Walkersville, MD, USA}
containing 10% human AB- sera, penicillin (100 U/mlj and streptomycin (loo ~.g/ml}, which served as th~ control medium.
8xperimental media were prepared by adding 25 ~Sl/ml of an appropriate aqueous dilution of vitaletheine V4 to obtain the following final concentrations in separate aliquots of medium containing cells otherwise identical with the controls: 0.1 fg/ml, 1 fg/ml, to fg/mI, 100 fg/ml, 1 pg/ml, and 10 pg/ml.
Purified cells (1 x 106) were seeded and incubated in the control and test media at 37°C under 5% COz. Gells were counted, and checked for viability daily by monitoring trypan blue (0.1% in phosphate riuffered saline) exclusion, and the media containing the same vitaletheine V,~ concentration were changed every two days to maintain physiological pH and to remove waste products from the cells.
Dramatic stabilization of the NK cell population in culture was observed with vitaletheine V,,. By day five, no cells survived in the unsupplemented, i.e., control medium. In media containing vitaletheine Va, 70 to 80% of the cells survived for more than a week. Although the extremes of the effective concentration were not defined in this particular experiment, two doses of vitaletheine Vd were selected for further study.

The results of the viability tests are summarized in Table I, following:
TABLB 1 ' ' DBV No V., 1 fa V.lml 1 fla y, 0 98 * 2 98 * 2 99 * 2 1 96 t 1.5 98 t 2 99 t 2.S

2 45 * 1.8 97 * 1.5 9B ~t 3 30 * 1.5 98 # 2.5 98 * 2 4 15 t 0.5 97 * 3 97 * 3 5-20 0 t 4 97 * 3 97 t 3 Vitaletheine V4 at concentrations of 1 fg/ml and 1 pg/ml stabilized between 70 and sod of the cells in culture for .
an entire month, at which time the cells were frozen for forthcoming functional studies. No cells remained in control v cultures, i.e., those lacking vitaletheine V," by day 6 oil the study. 'CJnlike the control cells whose ability to exclude trypan blue dropped precipitously from the first day in culture, 9713%
of the cells in the vitaletheine V4-supplemented media were viable after 3o days in culture, i.e., they excluded the dye.
EXAMPLE VII: Vitalet~e,~;te Modulato s Substitute for .
Er thro oietin Y p._....._.__ The early cell progenitors of recd blood cells in erythrapofesis (BFU-E), like the later erythroid progenitors (CFU-E), are dependent upon the presence of erythropoietin in liquid culture media to maintain their proliferative potential (Dessypris, E.N., and Krantz, S.B., 1984, ~. J'. Haemat~.. 56:
295-306, incorporated herein by reference).
Human bone marrow cells were obtained as surplus from experiments performed on material aspirated from normal volun tears with IRB approval and informed written consent. Peripheral blood cells were obtained from conunercially purchased buffy coats or surgical waste (umbilical curd blood). Mouse bone marrow was flushed from femurs and obtained as surplus: from experiments performed on C57B1/6 mice with animal committee approval. Human light density cells were separated by centrifugation over Ficoll-daitrizoate (sG 1.075) and depleted of adherent cells by incubatiori on serum coated plastic. Mouses cells were used without further fractionation. Cells were suspended at a concentration of 1 to 3 million cells per mi of Iscove~s medium (IMDM) supplemented with 10% heat-inactivated fetal calf serum (FCSj with varying concentrations of vitalethine or vitaletheine V4. One unit per ml of erythropoietin and medium without added factors sexved as positive and negative controls. =nitial incubations were carried out for 18 hours at 37°C. Cell suspen-sions were then pelleted and washed, and the cells were resus-pended in culture medium for plasma clot cultures similar to that previously described (Dessypris, E.N., Clark, D.A., McKee, L.C., and Kraritz, S.B., 1983, N. Ena~l. J. Med. 309: 69a-693, incorpo-rated herein by reference) except that fibrinogen was omitted, fetal calf serum replaced human (AB) serum, and human (AB) plasma replaced bovine plasma. The erythropaietin concentration for cultures of CFU-E was one unit per ml and for BFi1-E was 3 units per ml. Cultures were continued for the following periods: mouse and human cFU-E for two and seven days, respec-2o tively; and mouse and human BFU-E cultures for seven and fourteen days, respectively. Cultures were fixed, harvested, and stained for hemoglobin with benzidine, and scored as previously described (supra)»
Low concentrations of vitalethine (1 to 100 fg/ml) sustain the proliferative potential of BFU-8 initially deprived of erythropofetin (Figure 1a). Colony formation from human HFt~-E initially deprived of erythropoietin (lower square) are increased by vitalethine to levels (broken lines) initially containing erythropoietin, but lacking vitalethine (upper square). Colony formation from the early murine progenitors not exposed to vitalethine, and either initially exposed to or initially deprived of erythropoietin are represented by upper and lower triangles, respectively. vitalethine, depending upon concentration, either enhances or minimizes erythropoiesis from the CfL1-E progenitors (solid line). Although late erythroid progenitors are similarly affected by low concentrations of vitalethine, murine CFU-B are influenced more dramatically by higher concentrations of vitalethine; vitalethine from about 100 fg to 1 pg/ml minimizes colony farmation, while sstill higher concentrations (from 10 pg vitalethine/mI) enhance erythropoiesis (Figure .ta). Vitaletheine V4 (1Q0 fg/ml and higher concentra-5 tions) produces a stimulation of colony formaition from the CFU-~E
similar to that produced with high gg/ml concentrations of vitalethine (Figure l.a). Preformed vitaletheine V4 stimulates colony formation synergistically with erythrogoietin (Figure 1b) at much lower concentrations (from about l0 fg vitaletheine 10 V4/mly than the higher concentrations of vitalathine (from about 10 gg/ml) necessary for a similar response (3a). Bars are standard error of the mean.

EXAMPLE VIIx: xnfluenag~t~e~,~~ C,ai~,~ng ~n ,~~ablockina Reactions.
The procedure in Example IIA was followed except that magnesium or calcium ions were substituted for zinc ions in taaintaining the pH of the reaction. The use of calcitta~ or ainc cations resulted in benzyl derivatives of vitalethine, whereas the use of magnesium salts fn this procedure resulted in a cleavage of the N,N'-bis-carbobenzoxy-blocked beta-alethine (benzyl-V-8-S-V-benzyl; at the benzyl est~r bond, and the recovery of the corresponding cyclic urethane of Formula IIf.
The production of this cyclic urethane in the presence of magnesium ions was confirmed by IR analysis (unillustrated data), and by NMR analysis of the product of an intramolecular oondensa-tiori' of the two cyclic urethane moieties in DSO. The xearrange-ment reactions described herein are summarized as follows;
h ~ ~I=
brnsyl-V-8~8-V-brnsyi ~---> S brnsyi-V-~-I~-1-~~ brnsyl-W8-o-It (aq) -»s ..---..----> rulHry~t lodidr or rvliani~ acid or brnsyl drr3.wtivs !fq"jllCCl1 ~ C4++ 0r tllva/RaCN ' .
~-----~-~a cyclfo urrthrnr brnsy~~ drrivativa vv ~Csi~/~RSHj eondanration proeuct vitalrthrinr V, VSO3i --a5 + vssV tvs~sn)+wsH) (vitalrthinr) + (ytsSR) eyclio-nrrthan~
Intracellular concentrations of Mgt'" are mM, and Ca++
concentrations are known to range from, less than a~icromolar within the resting cell to ever nM in the plasma. ~,n viva, activities of the benzyl derivative and vitalethine were comparable as illustrated by Figures 22 and 23, a~ 17, 18, and 19, respectively.
~Doi, J.T., Luehr, G.W., and Musker, G1.K., ~'. ø,~r~. Chem. _~0;
5716-5719 (1985).
~Ballard, D.G.Ii., Hamford, C.Fi., and Weymouth, p.J., p~,oc. Rov.
Sor. Liebigs ,fin . Chem. 227: 155-183 (1954). Wxeland, von T., Lambert, R., Lang, fI.U., and Schramm, M., von G~., Justus Liebias Ann. Chemie 597: 181-195 (1955).
"'Albertson, N.F. , Organic Reactions, vol 12, John Wilay and Sons, Inc., New Yark, (1962), pp. 241-255. Fieser, L.F. and Fieser, M., en fo O a ' , John Wiley and Sons, Inc., New York, 1967, p. 1.00.
Similar theoretical rearrangements oaf other compounds to reportedly labile intermediates have been proposed by others, incorporated herein by reference as indicated. Substitution with the chemically similar nucleophiles (O, N, Nii, ~~r S) as described ' in Formula I are contemplated to produce analogous rearrangement products.
EXAMPLE IX: Theoretical Activat3o_p of A Be~l~yl Derivative Intracellular concentrations of Mg+''' are mM, and Ca++ .
concentrations are known to range from less than micromolar within the resting cell to over mM in the plasma. Since the calcium salt of a benzyl derivative (according to Example IIA and VII) and vitalethine have been shown to have similar biological activities and potencies {data not shown), activation of a benzyl derivative to a sulfenic acid of vitaletheine is theorized to involve the enzymic ionic pumps Within the cell.
EXAMPLE X: mherageutic Aonlicat~r~s of Vitalei-hine and Related Con~pnuryds in Neoplasia Cloudman S-91 murine melanoma cells (American Type Culture Collection #53.1, Clone M-3) from a (C X DBA)F1 male mouse were grown in 75 ml flasks (Corning Glass Works, Corning, New York, USA) containing Ham's F12 medium supplemented with 15~
fetal bovine serum, penicillin (l00 U/ml), and streptomycin (100 ~cgJml), all commercially available from Sigma Chemical Company, 3D St. Louis, MO, USA. Cultures were incubated at 37°C under 5.5~
carbon dioxide initially at 6 X 106 cells per ml far two days, with a medium change after one day. Cultures were then trypsin--izad, split into two fresh flasks at one half the cell density, above, and maintained for one week prier to injection in female (DBA X BALH c) mice (CD2F1/Hsd from &iarlan Sprague Dawley, Inc., Indianapolis, IN, tISA). For injection, cells were first trypsinized, washed 3 times in phosphate buffered saline, and diluted to I X 105 cel1sj10o ul phosphate buffered saline prior to subcutaneous injection on the rib cage.
The compounds were dissolved in water, filtered through an appropriate sterilizing filter (0.22~Cm non-pyrogenic, ~a Star L~fr"' from Costar. diluted to the desired concentration in sterile, physiological saline (0.1 ml), and injected 3 times per week intraperitoneally with a 27 gauge, 3j8 inch allergy syringe (8ecton Dickinson, Rutherford, NJ, USA); by gently lifting the skin on the abdomen and injecting horizontally, puncture of internal organs was avoided, thereby minimizing trauma to the mice.
Definition of several variables affecting tumor growth in these mice was necessary to establish confidence in the tumor model and conclusions therefrom derived. Fox instance, there was a significant difference in tumor development in old (14 weeJc, bottom curve, Figure 2) and young mice (4 week, top curve, Figure 1) injected With physiological saline. Although palpable tuneor development was significantly slower in the older anice than the young mice, gross metastasis in the lungs were as pronounoed if not mare evident in the older mice than in the younger mice.
These differences are postulated to be due to age-related differences in growth factors and immune surveillance. Except where specified, mice were matched for age to eliminate this complication in interpreting the results.
The compounds administered had to be pure to preclude complications resulting from trace contaminants; this was especially true when the contaminants wexe among the most potent of the compounds. f3-alethine, the immediate precursor of vitalethine in the phosgenation process, caused considerable stimulation of tumor development (Figure 3). Clearly, contami-nation of vitalethine with B-alethine could have abrogated any therapeutic effects of the former with the tumor promoting effects of the latter. Phosgenation removes the melanoma promoting properties of the B-alethine preparation almost completely; injections of 100 pg vitalethine/kg mouse produced none of the tumor promotion observed with the same injection dosage of 1i-alethine (Figure b) and, in fact, caused a non-signi-ficant decrease in tumor development (Figure 5). Even at injections of B-alethine one tenth that of vitalethine, signifi-cantly greater tumor stimulation was observed with the former than with the latter (Figuxe 6). Injections of vitalethine producing a response comparable to that observed with B-alethine were roughly one hundred times higher than tine latter (unillus-trated data), indicating that phosgenation resulted in at least l0 99% conversion of B-alethine to vitalethine. Small amounts of 8-alethine, resulting from incomplete conversion or from decomposition of the theoretically labile carboxy-amino group, could have been contaminating the preparation of vitalethine.
This Was of special concern since the tumor-promoting effects of 8-alethine were nearly saturated at 10 pg/kg mouse (Figures 7 and 8aj. Furthermore, the oscillation in the response of the tumors to increasing B-alethine concentrations could have made interpre-tations difficult (Figure. 8a). Fortunately, this neoplastic response was both reproducible (Figure 8a compared to Figure 8b) and interpretable, as described below.
As noted previously, the compounds are effective only at concentrations low with respect to most pharmacological compounds, This raises some interesting theoretical dilemmas.
One must assume that the compounds are not metabolised when administered at the low effective dosages, or one must try to retard the degradation of the compounds, for at these concen-trations there is essentially no metabolic reserve of the compounds. Clearly, the metabolism of every targeted cell and organ influences the outcome of these considerations; for instance, in sells capable of neither synthesising nor degrading the compounds, administration of the compounds alone produces the desired result; likewise, in cells in which, degradation and synthesis are favored, administration of only inhibitors of that degradation produces the desired result; in cells incapable of synthesizing the compounds but capable of degrading them, administration of both, inhibitors of their degradation and the compounds themselves, produce optimal results; similarly, in cells in which synthesis is favored but not degradation, no treatment is required. Conceptually, then, one must adjust the envirorunent of the cell to ensure a low steady state concentra-tion of the compounds, 5 The above considerations were necessary largely due to the propensity of compounds to polymerize when in the reduced or thiolate form. Indeed, when attempts were made to synthesize vitaletheine by irradiating high concentrations of its sulfenyl iodide with ultraviolet light, a polymer, vitaletheine V4, was 10 produced, along with about 15% of the material in a dimer form .
(presumably vitalethine resulting from autooxidation and reaction of the thiolate with the sulfenyl iodide to form disulfide) .
Vitaletheine v, was not without therapeutic potential, for as an analogue of vitaletheine and vitaleth3.ne, 3.t should inhibit .
15 degradation of the endogenous effectors. This potential was realized by combining vitaletheine VA and 8-alethine therapies note that the stimulation of tumor development by B~alethine (Figure sa) was offset by the preparation vitaletheine V4 (Figure 8b); and that the differences in these two surfaces (Figures 9, 20 10, and 11) define a therapeutic benefit of vi.taletheine V,~, especially at loo ng B-alethinelkg mouse. Unfortunately.
vitaletheine Vd as an analogue of vitaletheine and vitalethine also interfered with the function of the endogenous effectors (Figure 12). Some of this interference was removed by filtering 25 the vitaletheine V4 preparation through a sterile Miller-6V
filter (commercially available from Millipore Products Division, Bedford, MA, USA) and in so doing removing a large portion of the interfering analogue {figure 13). The di!lerences in these last two surfaces, Figures 22 and 13, indicated a therapeutic sub-30 stance in the vitaletheine v4 preparation permeable to the filter (Figures 14, 15, and 16). Figure 15 also depicted dose-independent differences in the two surfaces which were explained readily by the age differences between the mice in these two experiments, the younger mice developing tumor more rapidly than 35 the older mice, as in Figure 2. From these observations and arguments it was obvious that vitaletheine had to be administered in a form that was either extremely dilute ar stabilized to preclude polymerization. Since it was impractical to synthesize and characterize extremely dilute solutions of material, ways of presenting the cells or organisms with a stabilized form of vitaletheine were explored.
Problems with the synthesis and administration of vitaletheine were largely overcome by synthesis of vitalethine from the disulfide, D-alethine. Vitalethf.ne, lacking the thiolate moiety, did not polymerize, was easily diluted, and when administered was presumed reduced by endogenous thiols and t0 thiol-disulfide exchange mechanisms to vitale~theine. Further-more, vitalethine Was extremely potent in vivo, for unlike B-alethine and vitaletheine V4, vitalethine diluted to tumor development levels less than control values;; consequently an effective range of antineoplasic activity was indicated (Figure 17). The therapeutic window for vitalethine was even more striking when surface and curve apgroximations were attempted (Figures 18 and 19). I~ vivo reduction and polymerization was indicated by neoplastic responses of the tumor at injection concentrations above 100 pg vitalethine/kg mouse (Figure 18) ~0 which were very similar to the neoplastia responses to vitale-theine V4 (Figure 12), albeit the neoplastic responses to vitalethine occurred at much higher injection concentrations than thane for vitaletheine V4. The several explanations postulated for this observation include the following:
1) since a therapeutic response for vitalethine below 10o pg/kg mouse was strongly indicated by approximations using both a Xriging regional variable theory algorithm to analyze the entire study (Figures 17 and 18) and polynomial regression analysis of late points in the study (Figure 19), the neoplastia responses to vitaletheine V" formed from vitaletheine at injections concentrations less than 100 pg vi~talethine/kg mouse, were offset by therapeutic responses to remaining vitaletheine at these concentrations;
2) vitalethine itself interfered with vitaletheine effects when the concentration of the disulfide exceeds the reductive capacity at the target subcellular compartment; ' 3) the vitaletheine was formed intracellularly and was therefore partitioned within cellular and subcellular compart ments in the mice so that higher concentrations were achieved Without polyraerization, than can be achieved with the reduced compound 3n free solution; and 4) the vitalethine is contaminated with growth psvmoting precursors and metabolites.
Regardless of the interpretation of the neoplastic response, neoplastic development was significantly 7.ess in mice treated with vitalethine at injection.concentrations less than or equal to loo pgJkg mouse (Figure 19, bottom curve) , especially when compared to theoretical saturation profiles !or the stimu-lation o! tumor development by vitaletheine Vd (uppex curves), arid the theoretical development, viv , o! vitaletheine V~ from vitalethine via vitaletheine (middle curve). Combined therapies of vitalethine with other agents, and with inhibitors of vitalethine and vitaletheine degradation, especially vitaletheine V4 and/or b-alethine are also contemplated.
EXAMPLE XI: a t f a o a w yitalethine The experimental prooedure of Example X was repeated with the compounds, except the animals were inoculted in the flank.
When injected intraperitoneally thxee times per week (10 femtogram/kg mouse), beginning on the second day after tumor inoculation, vitalethine significantly diminishes tumor volume (~'70%) compared to controls reoeiving inoculations of tumor cells and injections of saline only (F3GS. 20 and 21). This regimen is predicated to these compounds, and which may play a role in the reported results, are induced 48 hours after chemical stinntlation. Vitalethine diminishes average tumor volume substantially over two ranges of concentrations as illustrated by responses to 10 fg/kg mouse (FIG. 20j and at 100 pg/kg mouse (FIG. 21). Data presented is calculated from measured tumor diameters, including mice with no tumor. Error bars indicate standard error of the mean.

$8 EXAMPLE XII: i Lysis of Human Leukemic Ce~~s i~i~56 Using a Preparation of Human NK (Natural Rillerl Ce s.
Glass non--adherent sells (GNAC's) were prepared as deasCribed in J.Exp died. 169: 99-113, 1989, incorporated herein by reference, using Ficoll-Hypaque gradients as described in Sc~~T~.Clin.Lab.~rtwe:gt. ~i,jsugp~,. x'71: 97-89, incorporated herein by reference, plating an glass, and passage through nylon wool columns as described in J.Immunol. 1'2: 420-423, incor-porated herein by reference. Tha targeted K562 human leukQmic cells (10,000) labeled with 5lCr (New England Nuclear Research Products, Dupont Company, Boston, MA, USA) as described in ~4rthrit,~,g ~egm. 2?: 1095-1100, incorporated herein by reference, were incubated in triplicate at each effector/target ratio (25, 12.5, or 6.25 times as many effector cells), or with 20~ Triton 7C-100, to determine cytotoxicity of the effector cells (GNAC's), or maximum lysis of the target cells, respectively, during a 4 hour incubation. Cytotoxicity of the GNAC's was determined initially and after 6 days of exposure to 1, 10, 10a, lOs, 104, lOs, 106 ag vitalethine/ml culture (1 through 7 in figure 23).
EXAMPLE XIII: Calcium Salt of Benx3rl Der xative Treats NS-1 Myeloma.
Instead of the direct measurement of the tumor diameter as in the melanoma model (Figure 2), myeloma development was estimated by an increase in the weights of mice (reflecting ascites .and solid tumor formation) relative to saline- and compaund~injected controls. Groups were normalized to the average weight of each group at the start of the study, and bars so are standard error of the mean.
NS-1 myeloma cells (ATCC TIE 18, P3/NS1/1-Ag4-1) were employed as inoculant in the BALBc/J mine model p these cells were about 90% effective in establishing myelamas in mice according to the exemplified procedure, and the untx-eated myelomas were substantially fatal within about two weeks. The cells were grown for several passages (preferably one week) in a sterile environ-ment consisting of RPMI 1640 (Whittaker M.A. Bioproducts, Walkersville, N07, usA) containing 10~ fetal calf serum (Hyclone Laboratories, Zogan, UT, USA), 2 mM L-glutamine, 5,000 units of penicillin, and 5 mg streptomycin in 75 cm1 polystyrene tissue-culture flasks (Corning Glassworks, Corning, NY, USA) in a humidified chamber at 37°C and under 6$ C~. To assure NS-1 propagation ~ vivo, it was essential to remove DMSO (the aryostatic agent dimethyl sulfoxide) through sevexal medium changes and dilutionsp this also served to maintain the cells in log-phase growth. Female BALBc/J mice were injected i.p. with 1o io° cells in 0.1 m1 of standard phosphate-buffered saline as soon as possible after weaning, transport, and indexing, as it has been found that the Ns-1 cell line employed does not generally perform optimally in animals which are mature or which have equilibrated with their environment. The mice were maintained with Wayne Rodent Blox (Wayne Research Animal Diets, Chicago, IL, USA) ad. lib. and tap water. Concentrations of the benzyl derivative of vitaletheine based upon the average body weight of each group of mice were injected f.p. in 0.1 ml sterile physio-logical saline starting the second day after tumor inoculation, and continuing every Monday, Wednesday, and Friday throughout the study. Weights of tumor-inoculated, compound-treated mice were significantly lower when treated with certain concentrations of the benzyl derivative (lower curve) compared to tumor-inoculated controls injected with saline (carrier) only (upper curve), and approximated those of saline-injected mice not challenged with tumor (middle curve) (Figure 24). The weight differences between drug-treated, tumor-inoculated mice and their corresponding drug-treated controls not challenged with tumor were dependent upon the concentration of the benzyl derivative (Figure 25).

Claims (47)

1. A compound of the formula:

wherein:
the set of double parentheses brackets the portion of the molecule bearing a charge p when z is 1; M1-(C-M)-M- (wherein C is the #2 carbon atom) is M1-(C=M)-M-, M1-(C-MA)-M-, or M1-(C-MA)-N-; and -(C=M)-M- (wherein C is the #5 carbon atom) is -(C=M)-M- or -(C-MA)=N-; Wherein A is X, -1, or a direct bond with the proviso that when -(C=M)-M- is -(C-MA)=N- or the compound is polymeric or internal cyclic or spirocyclic, A is optionally R; and M and M1 are as defined below; each R is independently H or a hydrocarbon radical;
X is a biologically-compatible cation or cationic complex; X' is a biologically-compatible ion or ionic complex;
M is S, O, N, or NH;
M1 is S or O with the proviso that M1, is also optionally N or NH when the compound is polymeric, or internal cyclic or spirocyclic;
Q is CR2 or a direct bond; Q1 is CR2, CR2CR2, or a direct bond;
Y is O, -[C=O]-R, or a direct bond;
a is the absolute value of ¦r/(r'+p+.SIGMA.s)¦ with the proviso that when (r'+p+.SIGMA.s) is >=O, at least one q or q' is zero, such that the sum of any charges on the remainder of the complex is balanced by charges on ion or ions, X or X', or ions, X and X'.
m is 0 or a whole integer from +1 to +5;
n is 1 or 2 when z is 1, and n is 1 or 1.5 when z is p is +1, 0, or -1;q and q' are each independently +1 or zero;
r and r' are each independently a whole integer from +1 to +4 or r' is a whole integer from -1 to -4;
s is -1 or O;y is 1 to 40;
z is +1 or +2; and wherein the compound has a molecular weight of no more than about 10,000 daltons.

2. A compound according to Claim 1 of the formula:

wherein Z W(0) is a neutral moiety associated with the compound of Claim 1, and w is a whole integer from +1 to +5.

3. A compound according to Claim 1 of the formula:

4. A compound according to Claim 2 of the formula:

5. A compound according to Claim 1, wherein y is from 1 to about 20.

6. A compound according to Claim 1, wherein the molecular weight of the compound is no more than about 5,000 daltons.

7. A compound according to Claim 4, wherein the molecular weight of the compound is no more than about 5, 000 daltons.

8. The compound of Claim 1, wherein the molecular weight is at least about 130 daltons.

9. A compound according to Claim 2 of the formula:

10. A compound according to Claim 9, wherein X is hydronium, H+, or Zn+2.

11. A compound according to Claim 10, wherein R is H.

12. A compound according to Claim 9 of the formula:

wherein n is 1 or 1.5.

13. A compound according to Claim 9 at the formula:

wherein n is 1 or 2, and Y is O.

14. Vitalethine or a physiologically-compatible salt or tautomer thereof.

15. Vitaletheine or a physiologically-compatible salt, tautomer, or polymer thereof.

16. A compound according to Claim 13, comprising a polymer of vitaletheine having four monomers.

17. An internal cyclic or spirocyclic form of a compound of Claim 2, of the following formula, produced by nucleophilic attack of at least one of the potentially nucleo-philic atoms (1,3,6); or at least one of the nucleophilic atoms S, or Y; or at least one of each in a monomer according to Claim 2 on at least one of its own doubly-bonded carbon atoms (2,5);
according to the following formula:

18. A compound according to Claim 32 of the formula:

wherein is , and q and a are zero; or is and a is -1.

19. An internal cyclic ox spirocyclic form of a compound according to Claim 4 of the following formula, produced by nucleophilic attack of at least one of the potentially nucleo-philic M atoms (1,3,6); at least one of the nucleophilic atoms S or Y; or at least one of each; in a monomer according to Claim 4 an at least one of its own doubly-bonded carbon atoms (2,5) or s, or both:

20. An internal cyclic or spirocyclic form of a compound according to Claim 39, wherein M and M1 axe O, produced by nucleophilic attack of at least one nucleophile O, S, or Y, of a monomer according to Claim 44 on at least one of its own carbonyl carbon atoms, or S, or both.

21. A polymer according to Claim 7, wherein y is greater than 1; wherein polymerization is initiated by nucleophilic attack of at least one of the atoms M1, M (3, 6), S, or Y of a first monomer of a compound of claim 4, on at least one of the doubly-banded carbon atoms (2,5) of at least one other monomer according to Claim 4; and wherein the monomers are of a different formula.

22. A polymer according to Claim 2 wherein y is greater than 1; wherein polymerisation is initiated by nucleophilic attack of at least one of the atoms M1, M (3,6), S, or Y of a first monomer of a compound of Claim 4, on at least one of the doubly-bonded carbon atoms (2,5) of at least one other monomer according to Claim 4; wherein the monomers are of the same formula.

23. A polymer according to Claim 9 wherein y is greater than 1; wherein polymerization is initiated by nucleophilic attack of at least one of the atoms O, S, or Y of a first monomer of a compound according to Claim 9, on at least one of the doubly-bonded carbon atoms (2,5) of at least one other monomer according to Claim 9 wherein y is 1.

24. A vitaletheine polymer of the formula:

wherein y is from about 2 to 40; R, X, X', q', r', Z, and r are as defined in Claim 1; and wherein (-)O-(C=O)-NH- is (-)-(C=O)-NH- and -(C=O)-NH- is -(C-O-)-NH-; or (-)O-(C-O)-NH- is (-)O-(C-O)-NH- and - (C=O)-NH- is -(C=O) -NH-; or both (-)O-(C=O)-NH-and -(C-O)-NH- are (-)O-(C-O-)-NH- and -(C-O-)-NH-, respectively.

25. The vitaletheine polymer of Claim 24, wherein y is from about 2 to 10.

26. The vitaletheine polymer of Claim 24, wherein y is from about 2 to 4.

27. A polymeric form of a compound wherein y is greater than 1, or a tautomer thereof, or an internal cyclic or spiro-cyclic form of a compound wherein y is 1, or a tautomer thereof, of the following formula produced by nucleophilic attack of at least one of the potentially nucleophilic atoms (1,3,6) or a nucleophilic atom S or Y of a monomer on at least one of the doubly-bonded carbon atoms (2,5) of at least one other monomer;
or on at least one of its own doubly-bonded carbon atoms (2,5), or S, or both:

wherein:
the set of double parentheses brackets the portion of the molecule bearing a charge p when z is 1; M-(C=M)-M- (wherein C is the #2 carbon atom) is M-(C=M)-M-, M=(C-MA) -M-, or M-(C-MA)=N-; and -(C=M)-M- (wherein C is the #5 carbon atom) is -(C=M)-M- or -(C-MA)=N-; wherein A is X, -1, a direct band, or R; and M is as defined below; each R is independently H or a hydrocarbon radical;
X is a biologically-compatible cation or cationic complex; X' is a biologically-compatible ion or ionic complex;
M is S, O, N, or NH;
Q is CR2 or a direct bond; Q1 is CR2, CR2CR2, or a direct bond;
Y is O, -[C=O)-R, or a direct bond;

a is the absolute value of ¦r/(r'+p+.SIGMA.s)¦ with the proviso that when (r'+p+.SIGMA.s) is >=0, at least one q or q' is zero, such that the sum of any charges on the remainder of the complex is balanced by charges on ion or ions, X ox X', or ions, X and X'.

m is 0 or a whole integer from +1 to +5;
n is 1 or 2 when z is 1, and n is 1 or 1.5 when z is 2;
p is +1, 0, or -1;q and q' are each independently +1 or zero;
r and r' are each independently a whole integer from +1 to +4 or r' is a whole integer from -1 to -4;
s is -1 or 0;y is 1 to 40;
z is +1 or +2; and wherein the compound has a molecular weight of no more than about 10,000 daltons.

28. A compound according to Claim 13, wherein is SOX'q, SX'q, SI, SI3, S2O3X', SH, or SOH.

29. A compound according to Claim 13, wherein is a thioester residue.

30. A cell culture medium including an effective amount of at least one of the compounds of Claims 1, 2, 4, 9, 14, 15, 17, 18, 19, 20, 21, 22, 26, 27, 28, or 29; and optionally further including an inhibitor of the metabolism of the compound or compounds present in the medium; or optionally further including an agent for enhancing cell propagation; or optionally further including both, an inhibitor of the metabolism and an agent for enhancing cell propagation.

31. In a method fax cell culture in vitro of the type wherein cells are cultured in an culture medium which at least supports cell life, the improvement evidenced by changes in cellular bioproductivity, function, or production) comprising incorporating in the medium an effective amount of at least one of the compounds of Claims 1, 2, 4, 9, 19, 15, 17, 18, 19, 20, 21, 22, 26, 27, 28, ar 29; and optionally further incorporating in the medium an inhibitor of the metabolism of the compound or compounds present in the medium; or optionally further incorpo-rating in the medium an agent for enhancing cell propagation; or optionally further incorporating in the medium both, an inhibitor of the metabolism and an agent for enhancing cell propagation.

32. A method for adapting resistant cells to culture, comprising culturing the resistant cells in a culture medium containing an adapting amount of at least one vitaletheine modulator according to Maims 1, 2, 4, 9, 14, 15, 17, 18, 19, 20, 21, 22, 26, 27, 28, or 29; and optionally further containing an inhibitor of the metabolism of the compound or compounds present in the medium; or optionally further containing an agent for enhancing cell propagation; or optionally further containing both, an inhibitor of the metabolism and an agent for enhancing cell propagation.

33. A method fox delaying cell senescence in vitro, comprising exposing cells under culture in a culture median to a senescence-delaying amount of at least one vitaletheine modulator according to Claims 1, 2, 4, 9, 14, 15, 17, 18, 19, 20, 21, 22, 26, 27, 28, or 29; and optionally further exposing the cells in the culture medium to an inhibitor of the metabolism of the compound or compounds present in the medium; or optionally further exposing the cells in the culture medium to an agent for enhancing sell propagation; or optionally further exposing the cells in the culture medium to both, an inhibitor of the metabolism and an agent for enhancing cell propagation.

34. A method for the synthesis of a vitaletheine modulator, comprising:
a) coupling a blocked A-alanine to N-hydroxy-succinimide to produce an active, soluble .beta.-alanine ester of N-hydroxysuccinimide;

b) coupling the active eater to the free amine of cystamine;
c) recovering blocked .beta.-alethine;
d) reacting the blocked .beta.-alethine with iodine and irradiating the product with UV light to selectively remove alkyl and aryl groups without removing the carboxyl moiety to produce the desired modulator; and e) stabilizing the modulators.

35. The polymer of Claim 22, wherein X is calcium or sine, y is 2, and the monomers are linked by Y.

36. A method for treating neoplasia comprising adminis-tering to an afflicted mammal an effective amount of a vitale-thins modulator of Claim 1 which is biologically active against the neoplasia, or which is activatable to said active modulator.

37. The method of Claim 36 wherein the modulator is of the formula:

wherein Z w(0) is a neutral moiety associated with the compound of Claim 1, and w is a whole integer from +1 to +5.

38. The method of Claim 36 wherein the modulator is of the formula:

39. The method of Claim 36, wherein y is from 1 to about 20.

40. The method of Claim 36, wherein the molecular weight of the modulator is no more than about 5,000 daltons.

41. The method of Claims, 36 or 37, wherein the vitalethine modulator is a modulator according to Claims 1, 2, 4, 9, 14, 15, 17, 18, 19, 20, 21, 22, 26, 27, 28 or 29.

42. A method for regulating cell function or bioproduc-tion comprising exposing a cell, in vitro or in vivo, to an amount of at least one vitaletheine modulator according to Claim 1 sufficient to normalize or improve function or bioproduction of the cell.

43. The method of Claim 42 wherein the cell is mammalian, including a human cell.

44. The method of claim 43 wherein the cell is an immunocyte or a cell infected with a parasite, pathogen, including a virus, and is exposed to the vitaletheine modulator or modulators in an amount sufficient to improve immunological surveillance or suppress the infection, or both.

45. The method of Claim 42 wherein the cell is exposed, in vitro, to from at least about 10 ag of vitaletheine modula-tor/ml cell culture or, in vivo, from at least about 100 ag vitaletheine modulator/kg weight of the organism.

46. A method for treating an immune disease or disorder, including an autoimmune ar immunodeficiency disease or disorder, in a mammal, including a human, comprising administer-ing to the mammal an amount of at least one vitaletheine modulator aocording to claims 1, 2, 4, 9, 14, 15, 17, 18, 19, 20, 21, 22, 26, 27, 28 or 29 sufficient to improve or normalise immunological function or bioproduction in the mammal.

47. The method of Claim 41 for improving or normalizing immunocyte function or bioproduction in a mammal, including a human, comprising extracting an immunocyte from the mammal, exposing the inmunocyte, in vitro, to sufficient vitaletheine modulator or modulators to stimulate immune function or biopro-duction thereof, and thereafter reintroducing the stimulate immunocyte into the afflicted mammal to treat the disease or disorder, including neoplasia or parasite- or pathogen-induced disease or disorder.