UA79753C2 - Mediators of hedgehog signaling pathways, compositions and uses related thereto - Google Patents

Abstract

The present invention makes available methods and reagents for inhibiting aberrant growth states resulting from hedgehog gain-of-function, ptc loss-of-function or smoothened gain-of-function comprising contacting the cell with a hedgehog antagonist, such as a small molecule, in a sufficient amount to aberrant growth state, e.g., to agonize a normal ptc pathway or antagonize smoothened or hedgehog activity.

Description

Description of the invention

Patterning is the process by which embryonic cells create a spatially ordered 2 arrangement of differentiated tissues. The physical complexity of higher organisms is formed during embryogenesis through the interaction of the differentiation line inherent in cells and the transmission of signals external to cells. Inductive interactions are essential in the embryonic pattern formation during the development of vertebrates, starting from the earliest foundation of the body plan and to the pattern formation of organ systems and to the period of generation of various cell types during tissue differentiation (|Oamidzop, E., (1990) 70 Oemeiyuortepi 108: 365 -389; Sigdop, 9. V., (1992) Seyi 68: 185-199; Oevzveyi, T. M. eyi ai., (1992) Seyi! 68: 257279|. The effects of the interaction of developing cells can be different . Typically, responding cells are switched from one cellular differentiation pathway to another by inducing cells, which differ from both the uninduced and induced states of responding cells. Sometimes cells induce their neighbors to differentiate in the same direction that is inherent itself (homeogenetic induction); in other cases, 12 the cell inhibits the differentiation of its neighbors in the same direction in which it differentiates itself. Cell interactions at the beginning of development can be sequential, and that the initial induction between the two cell types leads to a progressive increase in diversity. In addition, inductive interactions take place not only in embryos, but also in adult cells, and can establish and maintain morphogenetic patterns, as well as induce differentiation (U.V. Sygaop (1992) SeiI 68:185-199).

Members of the Neddeipod family of signaling molecules mediate many important short- and long-range processes in patterning during invertebrate and vertebrate development. In flies, a single peddepod gene regulates the patterning of segmental and imaginal discs. In contrast, in vertebrates, the peddeipod gene family is involved in the control of left-right asymmetry, polarity in the central nervous system, the control of somites and limbs, organogenesis, chondrogenesis, and spermatogenesis. with

The first Ppeddepod gene was identified by genetic screening in the fruit fly Drosophila Ge) melanogaster IMiisvivip-Moinaga, S. apa Muiesspatje, E. (1980) Maitsge 287, 795-801). This screening identified a number of mutations that affect embryonic and larval development. In 1992 and 1993 the molecular nature of the peddeiod (pp) gene of Drosophila |S.R., ee ei aI was reported. (1992), SeiI 71, 35-50), and since that time, several peddeiod homologues have been isolated from various vertebrate species. While only one peddepod gene was found in Drosophila and other invertebrates, numerous Neddepoa genes are present in vertebrates. yu

The Peddepyod family of genes in vertebrates contains at least 4 members, for example, paralogs of a single

Neddeiod of the Drosophila gene. Examples of genes and proteins of peddepods are described in (international RStT publications UUO in 95/18856 and MO 96/17924). Three of these members are known as JuOezegi peddeiod (OPP), Zopis peddepod (ZNAI) and Ipaiap Ge»)

Neddenpod (Ipy) are probably present in all vertebrates, including fish, birds and mammals. A fourth gene, known 39 as Todie-mipkieye Npeddepod (Ipp), is specific to fish. Yuezegi pPeddeiod (Opy) is expressed mainly in the testes, both during the development of the mouse embryo and in adult rodents and humans; Ipaiap peddeiod (II) is involved in bone development during embryogenesis and in adult bone formation; and the above-mentioned 5PY gene is primarily involved in morphogenetic and neuroinductive functions. Considering the important inductive role of peddepod polypeptides in the development and maintenance of vertebrate organs, the identification of proteins interacting with C

Peddenode, is the most essential and important, both for the clinic and for science. c Various Neddeipod proteins consist of a signal peptide, a highly conserved M-terminal region, and more

From" the divergent C-terminal domain. In addition to cleavage of the signal sequence in the secretory pathway |(G ee, 4.3. eg aY. (1992) Sei 71:33-50; Taraya, T. ey a). (1992) Sepez Yuemu. 2635-2645; Spapo, Yu.E. oh oh (1994)

OemeIuortepi 120:3339-3353), Neddeiod precursor proteins undergo internal autoproteolytic cleavage that depends on conserved sequences in the C-terminal part (I ee ei ei. (1994) Zsiepse et 266: 1528-1537; Rogieg ei a). (1995) Mayte 374: 363-366). This autocleavage results in a 19kDa M-terminal (se) peptide and a 26-28kDa C-terminal peptide (Ee et al. (1992), supra; Tabaille et al. (1992), supra; Spapd et al.). (1994), see above; Hey hey a). (1994) see above; Vytsgoi O.A., ei ai. (1995) Mine. sow Vio|. it. 15:2294-2303; Ropeg ey ay. (1995), see above; EKKeg, 5.S. her and her (1995) Sy. VioI. 5:944-955; Iai, S.). her sl 20 a). (1995) JuOemeIortepi 121:2349-2360). The indicated M-terminal peptide remains tightly bound to the membrane of the cells in which it was synthesized, while the C-terminal peptide is able to freely diffuse both into and out of the cell membrane. (1995) Maishge 374:363; eeeeeeeeeeeeeeeeeeeeeeeeeeee (1994), see above; Great things. (1995), see above; Maigi, E. ei ai. (1995) Yuemeyuortepi 121:2537-2547; KoeiipK, N. eyi a! (1995) Cell 81:445-455). Interestingly, retention of the M-terminal peptide at the cell membrane depends on autocleavage, whereas a truncated form of 29 NN, encoded by an RNA that ends exactly at the normal internal cleavage site, is able to diffuse

GF) ip o mygo (Ropeg ei a). (1995), see above and ip past |Ropeg, 9U.A. oh oh oh (1996) Seii/ 86, 21-34). Biochemical studies have shown that the autoproteolytic cleavage of the NN precursor protein proceeds through the formation of an intermediate internal thioester, which is then cleaved in a nucleophilic substitution reaction. Presumably, the nucleophile is a small lipophilic molecule that covalently binds to the end of the M-peptide from the 60 C-terminal side |Ropeg ey a). (1996), see above), binding it to the cell surface. The biological involvement is very deep. As a result of the specified binding on the surface of the producing cells

Neddepod, a high local concentration of the M-terminal Neddepod peptide is generated. It is this M-terminal peptide that is both necessary and sufficient for the near- and long-range Neddepod signaling activities in Drosophila and vertebrates. (1995), see above; EdKeg ei ai. (1995), see above; Hey, hey, hey. (1995), see above; because Wolf, N. ei a). (1995) SeiI 81: 445-455; Rogpeg ey a). (1996), see above: ГРией7. M.). her a). (1995) Sigt.

Vioi 5: 643-655 Rap, S.-M. oh oh oh (1995) Sei! 81:457-465; Mat, E. ei a). (1995) Maishtsge 375:322-325;

Ioreg-Mapyipe ei aiYi. (1995) Sy. Vio! 5:791-795; ShOiseg, 5. Seyi a). (1995) Yuemeiortepi 121: 2337-2347;

Eogrez, A. 4). her a. (1996) OemeIortepi 122:1125-1135).

NN is involved in the processes of formation of a pattern of near and far action in various sites during development

Drosophila. In somite polarity formation in the early embryo, it has a short-range effect that appears to be mediated directly, whereas in imaginal disc patterning it induces long-range effects through the induction of secondary signals.

Over the past few years, several Peddepyod genes have been cloned in vertebrates. Of these genes, the greatest 7/0 experimental attention has been paid to 5PY, because it is expressed in various organizing centers, which are the sources of signals that form neighboring tissues. Recent data indicate the involvement of ZNAI in these interactions.

The expression of Зай begins shortly after the start of gastrulation in the presumptive mesoderm, nodule - in |Snapoa ei ai mice. (1994), see above: Espeiaga, MU. ei ai). (1993) Se 75: 1417-1430), rats (Koeyipk, N. eb a). (1994) Sei 76: 761775) and chickens (|Kidaye, K. O. ei ai. (1993) Sei 75:1401-1416), and in the scutellum - in the zebrafish (striped zebra) (Ekkeg ei aiY. ( 1995), see above; Kgaizv, 5. ей аІ. (1993) SeiI 75: 1431-1444).

In the chicken embryo, the expression pattern of ZNy in the nodule develops left-right asymmetry, which is probably responsible for the location of the heart on the relative left and right sides | emip, M. ei a!. (1995) Sei! 82: 803-814).

In CNO, BP from the chorda and basal lamina probably induces the ventral cell tract. Ectopic expression of PP leads to ventralization of large areas of the midbrain and hindbrain in the mouse (Espeiaga et al. (1993), see above; Sooargisp, H. M. ek a! (1996) Sepez Oeu. 10:301-3121, frogs (Heporiz) | KoeiipK, N. ei ai. (1994), see above; Kii and Amara , A. ei a). (1995) Mine. SeiYi Meygovsi. 6:106-121), and striped zebra

IEIdseg ei aiYi. (1995), see above; | (gaez ei aY. (1993), see above; Nattegzsptisi, M., ei ai. (1996) Sepev

OEM 10:647-658). In explants of the intermediate neuroectoderm at the levels of the spinal cord, the ZPP protein induces the development of the basal lamina and motor (motor) neurons at different limiting concentrations: the development of the basal lamina at high concentrations and motor neurons at lower concentrations. (1995), see above; My, my, my, my. (1995), see above; Tapare, U. eyi a). (1995) Sigt. Vio 5:651-658). In addition, the blocking of i) by antibodies suggests that Z5Py, which is produced by the notochord, is necessary for notochord-mediated induction of the motor neuron tract |Magy ef aY. (1995) see above) Thus, the high concentration of ZYi on the surface of cells of the midline producing ZPYy is probably responsible for

Contact-mediated induction of the bottom plate, which is observed in mygo | Riasgek, M. ei ai. (1993)

OemeIortepi 117:205-218)Y, and for the location of the bottom plate along the middle line directly above the chord and past it. Lower concentrations of Zn released from the chorda and basal lamina presumably induce motor neurons in more distant ventrolateral areas in a process that has been shown to be contact-independent and migo (Matada, T. eyi aiyi. (1993) Seii/ Y 73:673-686). In explants taken at the level of the midbrain and forebrain, ZPy also induces the corresponding ventrolateral types of nerve cells, dopaminergic (Neupez, M. BE aiYi. (1995) Metsygop 15: 35-44; MUapud, M. 7. ei a). (1995) Have May. 1: 1184-1188) and cholinergic (Egiswop, 9. ei ai. (1995) Seii/ 81: 747-756 | precursors, respectively, indicating that the ZNPy is a general inducer of ventral directionality throughout the length of the CNS. These observations raise the question of how the differentiated response to the ZN is regulated in specific "antero-posterior positions. in the Zn from the midline, it also forms a pattern of the paraxial regions of the vertebrate embryo, somites in the body. (BRap ei aiY. (1995), see above| and mesenchyme of the head rostral to the somites (Nattegestpdi ei aiYi. (1996), see i?" above). In explants of paraxial mesoderm of diseased chicks and mice, ZAy stimulates the expression of specific sclerotome markers, such as Rahi and Tmivi, at the expense of the dermomyotome marker RahZ3. More moreover, experiments with a filter barrier indicate that ZPy mediates the induction of sclerotome -I directly, and not through the activation of a secondary signaling mechanism (Bap, S.-M. apa Tezvzieg-I amidpe M. (1994) Seiyi 79,1175 -1186). induces myotomal gene expression | (1996), see above; Shdoppzop, K. Y. ei ai. -I (1994) Se 79:1165-1173: Miipviegregu, A. E. ei ai. (1995) Sepez Yuem. 9: 2911-2922; MUeipregu, E. 5. ei ai. (1996). (1995), see above). It is confusing 4) that higher concentrations of ZNAI are required for myotome induction in chickens than for the induction of sclerotome markers |Mypsiegregd ei a). (1995) see above); although the sclerotome originates from somite cells located much closer to the chord. Similar results were obtained in zebrafish, where high concentrations of two peddenodes induce the expression of genes of myotome markers and inhibit the expression of sclerotome markers. (1996), see above). However, in contrast to amniotes, these observations are consistent with the structure of the fish embryo,

F) because here the myotome is the dominant and more axially located component of the somites. Thus, the modulation of ZPP signal transmission and the acquisition of new signaling factors can modify the structure of somites during the development of vertebrates. In vertebrate limb buds, a subtype of posterior mesenchymal cells, the "Zone of polarizing activity" (ZPA), regulate digit identity in the anteroposterior direction (for a review, see Nopid, I. 5. (1981) Mayte 291: 72-13). Ectopic expression of ZHy or application of granules coated with ZAI peptides produces an effect similar to the effect of grafts of the posterior ZPA, generating mirror-image-like doubling of fingers (Spapa et al. (1994), see above; Gore Magipel et al.). (1995), see above; Kiadie e( aiii. (1993), see above) (Fig. 29). 65 Thus, finger identity appears to depend primarily on 5p concentration, although it is possible that other signals may transmit this information over substantial distances, which is apparently required for AP

(antero-posterior) pattern formation (100-15Ωm). Similarly, the interaction of NN and ORP in imaginal disks

Drosophila, ZNIpy in limb buds in vertebrates activates the expression of Btr2, a homologue of arr (Egapsiz, R.N. ei ai. (1994) OemeIortepi 120:209218). However, unlike ORR in Drosophila, Vtr2 was unable to act like the polarizing effect of ZI, upon ectopic application in the limb bud of chicks |Rhaps ei ai. (1994), see above). In addition to the antero-posterior formation of the pattern, ZAi is also probably involved in the regulation of limb growth in the proximodistal direction by inducing the synthesis of fibroblast growth factor (ESE4) in the posterior apical ectodermal crest (I asheg, E. ey aiYi. (1994) SeiI 79: 993-1003; Mizul"apaeg, |. ei aI. (1994) Maitsge 371:609-612)I. 70 The close relationship between Neddepyod proteins and WMR appears to be preserved in many, but perhaps not all, sites of expression of Neddepod in vertebrates. For example, it was shown that in the hind intestine of chickens, ZNAI induces the expression of Btr4, another homologue of drr in vertebrates |Kobegiv, Yu. 9. ei A. (1995) YuOemeiortepi 121: 3163-3174).

Moreover, ZPy and Btr 2, 4, or 6 show a striking correlation of their expression in epithelial and mesenchymal cells of the stomach, genitourinary system, lungs, tooth buds, and hair follicles |VidosoaYa, M. 9. apa

MeMapop, A. R. (1995) Hume. Vioi 172: 126-168). Next, Iyy, one of the other two mouse Ppediod genes, is expressed adjacent to Wtr-expressing cells in the developing intestine and cartilage |Vidosa apa

MeMapyop (1995), see above).

In the process of cartilage formation, chondrocytes pass from a proliferative state through an intermediate state preceding the hypertrophic state to differentiated hypertrophic chondrocytes. IPI is expressed in chondrocytes,

Which are in a state that precedes the hypertrophic state and initiates a signaling cascade that leads to the blockade of chondrocyte differentiation. The direct target of this process is the perichondrium around the domain of Ipy expression, which corresponds to the expression of OH and Raisney (Rhys), conserved transcriptional targets of Neddepod signals (see below). Presumably, this leads to a secondary signal transmission, resulting in the synthesis of a protein related to parathyroid hormone (PTHRH) in the periarticular perichondrium. RTNHR itself gives a reverse signal to chondrocytes that are in a previous hypertrophic state, blocking their further differentiation. At the same time, RTNHR suppresses the expression of Iy, thus forming a negative i) feedback loop that modulates the rate of differentiation of chondrocytes.

Raisney was originally identified in Drosophila as a somite polarity gene, one of a group of developmental genes affecting cell differentiation within individual somites, which occurs in a homologous row along the s z z posteroanterior axis of the embryo. | See Nooreg, U. E. ey aiYi. (1989) Sci 59: 751; and Makapo, U. ei ai. (1989)

Maiige 341:508). Expression patterns of the raisned homologue in vertebrates suggest its involvement in the development of the neural tube, skeleton, limbs, craniofacial structure, and skin. M

Genetic and functional studies demonstrate that Raisped is part of the peddepod signaling cascade, an evolutionarily conserved pathway that regulates the expression of a large number of downstream genes. | See me)

Regitop, M. (1995) Se 80:517; and Regpitop, M. (1996) Se 86:513). Raisney participates in constitutive and transcriptional repression of target genes; this action is opposed by a glycoprotein encoded by the peddeiod gene, or its homologue in vertebrates, which induces transcriptional activation. Genes under the control of this pathway include members of the MUpi and TOR-beta families.

Raisned proteins have two large extracellular domains, twelve transmembrane segments and several "cytoplasmic segments". above; dopvop, MK Yi. ei aiYi. (1996) Zsiepse 272:1668; and Knight, N. er - s aI. (1996) Science 85:841). The biochemical role of raisned in the peddepyod signaling pathway is unclear. However, direct interaction with the peddepod protein was reported | Spep, U. eyi a. (1996) SeiI 87:553), and also that raisped can;" participate in the peddepod receptor complex together with another transmembrane protein encoded by the ztooiPpepead gene (|Regititop, see above; and Spep, see above).

The raisped homolog in humans was recently cloned and mapped on chromosome 9422.3 Juoppsop, see above; and Napp, -And see Above). This refers to basal cell nevus syndrome (BNS), which is characterized by developmental abnormalities that include rib and craniofacial changes, hand and foot abnormalities, and spina bifida. ik SBCN also contributes to many types of tumors, and the most common is basal cell carcinoma-I (BCC), which is located in many places on the body and appears during the first two decades of life.

However, most cases of BCC are not associated with the mentioned syndrome and occur sporadically in small amounts 1 in sun-exposed areas in middle-aged or elderly people of Northern European origin. 4) Recent studies of SECN-related and sporadic BCC suggest that functional loss of both raisned alleles leads to the development of Schoppzop BCC, see above; Napp, see above; and SaiMPapi, M. K. ei ai. (1996) Maiige Sepeiyisvz 14:78)|. Single deletions of chromosome 9422.3 alleles occur frequently in both sporadic and hereditary BCC. Linkage analysis revealed that in tumors from patients with SBCN, the defective inherited allele was preserved, while the normal allele was lost.

F) Sporadic tumors also showed loss of both functional alleles of raisped. Of the twelve tumors in which Raisned mutations were identified by single-stranded conformational polymorphism screening, nine had a chromosomal deletion of the second allele, and the other three had inactivating mutations in both 6o alleles. above). These mutations did not occur in the corresponding DNA of germline cells.

Most of the identified mutations resulted in premature stop codons or frameshifts (epsy, M. 9. ei aY.,, Nyt. Sepei. 1997 Axis 100 (5-6):497-5021. Some, however, were point by mutations leading to amino acid substitutions in both the extracellular and cytoplasmic domains.These mutation sites may show the functional importance of interactions with extracellular proteins or with cytoplasmic 65 downstream participants in the signaling pathway.

The inclusion of raisnea in the inhibition of gene expression and frequent allelic mutations of raisnea in BCC confirm the suppressive function of this gene in relation to tumors. Its role in the regulation of gene families known to be involved in cell signaling and cell-cell interactions suggests a possible mechanism of tumor suppression.

The present invention provides methods and agents for inhibiting the activation of the peddeiod signaling pathway, for example, to inhibit abnormal growth conditions that result from phenotypes such as ris loss-of-function (oz8-oY-Tipsiop), Neddeiod gain-of-function (daip-oi-Tipsiop) or the acquisition of function by which the cell is brought into contact with an agent, such as a small molecule, in an amount sufficient to stimulate normal ris activity, inhibit normal peddeiod activity, or inhibit /o neped activity, e.g., to reverse or control an abnormal growth state .

Figures 1-31 show reactions for the synthesis of compounds according to the proposed invention.

Figures 32a-32| illustrate representative compounds according to the present invention.

Figure 33 shows the effect of compounds according to the invention on peddeipod signaling in human basal cell carcinoma (BCC).

Figure 34 illustrates the effect of compounds according to the invention on apoptosis in BCC tissue.

Figure 35 depicts the effect of compounds according to the invention on pediode signaling in cells 5-12 and

Mt-29.

Figure 36 depicts the effect of compounds according to the invention on peddepod signaling in PO-3 cells and

Kt-4.

Figure 37 presents sequences of probes and primers used to determine Peddepod activity in cells.

Figures 38 and 40 illustrate the characteristic effect of compounds according to the invention on the expression of 01 1-1 and

SARON in ris-null cells.

Figures 39 and 41 show the characteristic effect of the compounds according to the invention on the expression (1 I-1 and ZARON cells) in NERM cells.

Best examples of implementation of the invention i)

I. Review

The present invention relates to establishing that signal transduction pathways that are regulated by peddepod, raisnea (ris), action and/or ztooiPeped can be inhibited, at least partially, by small molecules. Without resorting to the limitations of the framework of the exact theory, the activation of the receptor can be considered as the mechanism by which these agents act.

For example, the ability of these agents to inhibit the proliferation of cells with a loss of raisind function (ris 9) may be based on the ability of such molecules to interact with Neddepod, raisind, or ztoepipeid, or at least interfere with the ability of these proteins to activate the signal transduction pathway mediated by Ppeddens, ris and /or ztooipepead. b

Thus, it is hypothesized that these small molecules that interfere in some aspects with the signal transduction activity of pedeiod, ris, or ztoipepei will also be able to inhibit proliferation (or other biological effects) in normal cells and/or cells with a raisned loss-of-function phenotype , the phenotype of the acquisition of the pedeiod function or the phenotype of the acquisition of the ztooipepeid function. Thus, it is suggested that in some embodiments, these compounds may be useful for inhibiting peddepod activity in normal cells, that is, those that do not have a genetic mutation that activates the peddepod pathway. In the best embodiments, the proposed inhibitors are - c organic molecules having a molecular weight of less than 2500 atomic mass units, better less than 1500 atomic c mass units, and even better less than 750 atomic mass units, and capable of inhibiting at least some biological "" activities of peddepod- proteins, mostly specifically in target cells.

Thus, the methods of the proposed invention include the use of small molecules that stimulate the inhibition of ris in pedapode signal transduction, for example by inhibiting the activation of stoepipei -I or components "downstream" in this signaling pathway, in the regulation of restoration and/ or the functional activity of a wide range of cells, tissues and organs, including normal cells, tissues and organs, as well as those with loss-of-function phenotypes, gain-of-function of peddepods, or gain-of-function of pedodes. For example, the proposed method has therapeutic and cosmetic options for use in the regulation of nerve tissues, cysts and cartilage, in the regulation of spermatogenesis, regulation of smooth muscles, regulation of the lungs, liver and other organs that develop from the primary intestine, regulation of hematopoietic functions, regulation of skin and hair growth, and so on. In addition, the proposed method can be carried out on cells presented in culture (ip migo) or on cells in the animal body (ip mimo). |See, for example, publications PCT UUO 95/18856 and UMO 96/17924 (descriptions of which inventions are incorporated herein by reference)|.

In a preferred embodiment, the proposed method can be applied to the treatment of epithelial cells having a phenotype of loss of function of ris, gain of function of peddepod or acquisition of function of pedodepod. Example,

F, the proposed method can be used in the treatment or prevention of basal cell carcinoma or other disorders associated with the peddepod pathway.

In some embodiments, the proposed antagonist can inhibit the activation of the peddepod pathway by binding to boron 8tooiyepei. In some embodiments, this antagonist can inhibit activation of the peddepod pathway by binding to Raisnea.

In another preferred embodiment, the proposed method can be used as part of a treatment regimen for malignant medulloblastoma and other malignant primary neuroectodermal tumors of the CNS.

According to another aspect of the present invention, there are provided pharmaceutical preparations containing as an active ingredient a peddenode antagonist, a ris agonist, or a ztoiyepei antagonist, as described herein, administered in an amount sufficient to inhibit ip mimo proliferation or other biological consequences of loss of ris function, acquisition neddepod functions or acquisition of stooiNepea functions.

The proposed methods of treatment using pedeioad antagonists, raisinide agonists, or ztooipepey antagonists may be effective in both human subjects and animal subjects. Animal entities include pets and livestock that are kept either as pets or for commercial purposes.

For example, dogs, cats, cows and oxen, horses, sheep, pigs and goats.

I. Definition

For convenience, definitions of terms used in the specification, examples, and attached patent claims are provided here. 70 The expression "aberrant modification or mutation" of a gene refers to such damage to the gene as, for example, the removal, replacement or introduction of nucleotides into genes, as well as to significant chromosomal rearrangements of the gene and/or incorrect methylation of the gene. Similarly, the expression "misexpression of a gene" refers to aberrant levels of transcription of a gene relative to those levels in a normal cell under similar conditions, as does splicing

The mRNA is not the wild type of the transcribed gene. "Basal cell carcinomas" exist in a variety of clinical and histological forms, such as forms of ulcerative nodular, superficial, pigmented, scleroderma, fibroepithelioma and nevoid syndromes. Basal cell carcinomas are the most common skin neoplasms found in humans. Most new cases of non-melanoma skin cancer fall into this category.

The term "burn wounds" refers to cases where large areas of the skin's surface have been removed or

Lost by humans under the influence of heat and/or chemicals.

The term "carcinoma" refers to a malignant neoplasm consisting of epithelial cells that have a tendency to infiltrate neighboring tissues and give rise to metastasis. Examples of carcinomas include: "basal cell carcinoma," which is an epithelial tumor of the skin that, although rarely metastasizes, has the potential for local invasion and destruction; "squamous cell carcinoma", which generally refers to carcinomas that arise from squamous epithelium and have cubic cells; "carcinosarcoma", which is a malignant tumor consisting of cancerous and sarcoma tissues; "adenocystic carcinoma" - a carcinoma that i) is characterized by hyaline cylinders or bands or mucoid stroma, separated or surrounded by nests or cords of small epithelial cells arising from the chest and salivary glands and mucous glands of the respiratory tract; "epidermoid carcinoma", which refers to malignant cells that have a tendency to differentiate as epidermal cells, that is, they have a tendency to form pinnate cells and undergo keratinization; "nasopharyngeal carcinoma", which refers to malignant tumors that arise in the epithelial lining of the posterior part of the nasal cavity; and "renal cell carcinoma," which refers to carcinoma of the renal parenchyma composed of tubular cells of various types. Other cancerous epithelial tumors are "papillomas", which refer to soft tumors derived from the epithelium and which have the papillomavirus as the causative factor (causing agent); and "epidermoid," referring to a brain or meningeal tumor formed by the inclusion of ectodermal elements during closure of the neural tube.

The term "corium" or "dermis" refers to the layer of skin in the epidermis, which consists of tough vascular-fibrous connective tissue, and contains nerve endings and touch receptors. Root hairs, sebaceous glands, and sweat glands are epidermal structures that are located in the skin. "

The term "dental tissue" refers to tissue in the mouth that is similar to epithelial tissue, such as gum tissue. The proposed method of the invention is suitable for the treatment of periodontitis. "Dermal skin ulcers" refer to skin lesions caused by superficial tissue loss, usually with inflammation. Dermal skin ulcers that can be treated by the method of the present invention include bedsores, diabetic ulcers, varicose ulcers, and atheromatous ulcers. Prolonged bed sores are chronic ulcers that are the result of pressure applied to areas of the skin -I for significant periods of time. Wounds of this type are often called pressure sores or bedsores. Varicose ulcers are the result of stagnation of blood or other fluids in defective veins. Atheromatous ulcers refer to necrotic skin in the area around arteries with poor blood flow. - and "EDVO" is an effective dose of the drug, which provides 50 90 of its maximum response or effect.

The term "effective amount", e.g., of a Npediode antagonist according to the proposed method of treatment, refers to such an amount of antagonist in the preparation that, when used as part of the desired 4) dosage regimen, causes, e.g., a change in the rate of cell proliferation and/or the state of cell differentiation and/or the level of cell survival in accordance with clinically acceptable standards of disease treatment or use for cosmetic purposes.

The terms "epithelium", "epithelial" and "epithelioma" refer to the cellular covering of the internal and external surfaces of the body (skin, mucosa and serosal), including glands and other structures, for example,

F) corneal, esophageal, epidermal cells and epithelial cells of hair follicles. Another exemplary ka epithelial tissue is the olfactory epithelium-I pp, which is a pseudostratified epithelium that lines the olfactory region of the nasal cavity and contains olfactory receptors; glandular epithelium, which refers to the epithelium, because it is composed of secretory cells; squamous epithelium, which is an epithelium composed of flattened squamous cells. The term epithelium may also refer to transitional epithelium, such as that lining hollow organs, which is subject to significant mechanical changes of contraction and stretching, for example, tissue representing the transition between stratified squamous and columnar epithelium.

The term "epithelialization" refers to healing by the growth of epithelial tissue on a bare surface. 65 The term "epidermal gland" refers to a cluster of cells associated with the epidermis and specialized for the secretion and excretion of substances unrelated to their normal metabolic needs. Example,

"sebaceous glands" are holocrine glands in the corium that secrete subcutaneous fat and sebum. The term "sweat glands" refers to sweat-secreting glands that are located in the corium or superficial tissue and open through a duct on the surface of the body.

The term "epidermis" refers to the most superficial and avascular layer of the skin, derived from the embryonic ectoderm, the thickness of which varies from 0 to 1.4 mm. On the palmar and plantar surfaces, it contains, in the direction from the inside to the outside, five layers: the basal (germ) layer, consisting of rods and cones, located perpendicularly; spinous or intermediate layer consisting of flattened polyhedral cells with short processes or spines; granular layer consisting of flattened 7/0 Granular cells; shiny (transparent layer), which consists of several layers of shiny, transparent cells, the nuclei of which are indistinct or absent at all; and the stratum corneum, which consists of flattened, keratinized anucleate cells. In the epidermis of the main surface of the body, a transparent layer is usually absent. "Excisional wounds" involve tears, scratches, cuts, punctures, or tears in the epithelial layer of the skin and may extend into the dermal layer and even into the subcutaneous fat and beyond. Excisional wounds can be /5 the result of surgical procedures or accidental penetration of the skin. "Growth state" of a cell refers to the rate of proliferation of the cell and/or the state of differentiation of the cell. An "alternative growth state" is a state of growth characterized by an abnormal rate of proliferation, for example, a cell shows increased or decreased rate of proliferation compared to a normal cell.

The term "hair" refers to a filamentous structure, in particular to a specialized epidermal structure consisting of keratin, which develops from the hair papilla in the corium of mammals and is unique to this group of animals. In addition, the term "hair" can be applied to the aggregate of such hair. The term "hair follicle" refers to the tubular invaginations of the epidermis that surround the hair and from which the hair grows. The term "epithelial cells of the hair follicle" refers to the epithelial cells surrounding the dermal papilla in the hair follicle, such as stem cells, outer root sheath cells, matrix cells, and inner root sheath cells. Such cells can be normal benign i) cells or transformed/immortalized cells.

The term "neddeipod antagonist" refers to a substance that enhances or recapitulates the biological activity of a gene, as well as represses the transcription of target genes. Predominantly, Ppeddepod antagonists can be used to overcome the loss of ris function or the acquisition of ztooipeped function, the latter also refers to ztooipeped antagonists. The term "peddepod antagonist" as used herein refers not only to any substance that can act by directly inhibiting the normal function of the peddepod M protein, but also to any substance that inhibits the peddepod signaling pathway and thereby recapitulates the function of fig. me)

The term "gain of function pPeddeiod (peddepod daip-oi-Tipsiop)" refers to an aberrant modification or mutation of a ris gene, a Neddepod gene, or a ztooiNepeai gene, or to an increased (or decreased) level of expression of such a gene, resulting in a phenotype that is cell-like peddepod protein, for example, with abnormal activation of the peddepod signaling pathway. Gain of function may include the loss of the growth gene product's ability to regulate the expression level of C genes, for example, C1, C1, and C13. The term 'peddepod gain-of-function' is also used here to refer to any similar cell phenotype with c (eg, one that exhibits excessive proliferation) that originates through a change somewhere in the signal transduction pathway, including but not limited to limiting, modifications or mutations of the peddeps itself. For example, a tumor cell with an abnormally high rate of proliferation due to activation of the Ppeddepod signaling pathway would have a gain-of-function phenotype of non-dedepod, even if peddepod is not mutated in such

Cells -And As used herein, the term "immortalized cells" refers to cells that have been altered by chemicals and/or recombinant drugs so that they have the ability to grow through an unspecified number of divisions in culture. -I The term "inner epithelial tissue" refers to body tissue that has properties similar to the epidermal layer of the skin. As an example, we can name the lining of the intestines. The method according to the proposed invention can be used to accelerate the healing of certain internal wounds, for example, 4) wounds resulting from surgical intervention.

The term "keratosis" refers to a proliferative skin disease characterized by hyperplasia of the stratum corneum of the epidermis. Examples of keratosis include follicular keratosis, palmar and plantar keratosis, laryngeal keratosis, lichen planus, and actinic keratosis.

The term "I Or" means the dose of the drug that is lethal for 5095 subjects of the study.

F) The term "nail" refers to the horny skin plate on the dorsal surface of the distal end of a finger or toe.

The term loss of function of risney "raisney Iov5v-oY-Tipsiop)" refers to an aberrant modification or mutation of the ris gene, or to a reduced level of gene expression, resulting in a phenotype that is similar to the cellular Npeddepod protein, for example, with aberrant activation of signaling path of peddepod. Loss of function may include the loss of the ability of the gene product to regulate the level of expression of C genes, for example, CII1, O1i2 and

SIiIZ. The term "loss of ris function" is also used herein to refer to any cell-like phenotype (eg, one that exhibits excessive proliferation) that results from an alteration 65 anywhere in the pediod signal transduction pathway, including but not limited to , modifications or mutations of the growth itself. For example, a tumor cell with an abnormally high rate of proliferation due to activation of the peddepod signaling pathway would have a "ris loss of function" phenotype, even if ris is not mutated in such a cell.

The term "patient" or "subject" should be considered as the subject of the method, meaning both a human and any other animal.

The term "prodrug" includes compounds that, under physiological conditions, are converted into therapeutically active substances according to the proposed invention. The general method of preparation of the drug involves the selection of components that are hydrolyzed under physiological conditions to obtain the desired molecule. In other embodiments, the prodrug is transformed by the enzymatic activity of the host animal.

As used herein, the terms "proliferation" and "proliferative" refer to cells that are in the process of mitosis.

Throughout this application, the term "proliferative skin disease" refers to any disease/disorder of the skin characterized by unwanted or aberrant proliferation of skin tissue. These disorders are typically characterized by epidermal cell proliferation or incomplete cell differentiation and include, for example,

X-linked ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic /5 piperkeratosis and seborrheic dermatitis. For example, epidermodysplasia is a form of defective development of the epidermis.

Another example is "epidermolysis", characterized by a weakened state of the epidermis with the formation of bubbles and bullae spontaneously or as a result of trauma.

As used herein, the term "psoriasis" refers to a hyperproliferative skin disease that alters the regulatory mechanisms of the skin. In particular, disorders are formed that cause primary and secondary changes in epidermal proliferation, inflammatory reactions of the skin and the expression of regulatory molecules, such as lymphokines, and inflammatory factors. The skin of a patient with psoriasis is morphologically characterized by an increased turnover of epidermal cells, thickened epidermis, abnormal keratinization, infiltration of inflammatory cells into the dermis layer and penetration of polymorphonuclear leukocytes into the epidermal layer, which leads to an increase in the duration of the basal cell cycle. In addition, hyperkeratotic and parakeratotic cells are present. high school

The term "skin" refers to the outer protective covering of the body, consisting of the corium and epidermis, and which, of course, contains sweat and sebaceous glands, as well as follicular structures. Everywhere in this application i) the adjective "leathery" may be used, and it is quite clear that it refers to features of the skin in general, according to the context in which it is used.

The term "gain-of-function of ztoipepesy" refers to an aberrant modification or mutation of the zto gene, or to an increased level of gene expression, resulting in a phenotype that is similar to a peddepod protein cell, for example, with impaired activation of the peddepod signaling pathway. Without wishing to be bound by any particular theory, we note that ris may not signal directly into the cell, but rather M interacts with ztooipeped, another protein located in the peddeipod signaling pathway downstream of ris |Magido ei ai., (1996)

Maishige 384: 177-179). This gene is the segment polarity gene, necessary for the correct formation of each me)

335 of the Drosophila segment (AiYsedo ei ai., (1996) SeiI 86: 221-232). Human homologues of zto were identified. See, for example, Ziope eyi a). (1996) Mayte 384:129-134, and SepVapkK assayviop 84401). The ztooiyepei gene encodes an integral membrane protein with the characteristics of receptors of heterotrimeric protein b, i.e. transmembrane family 7TM. This protein shows homology to Drosophila Rgig2g7Ie (E2), a member of the wingless variety. At first, it was thought that zto encodes a receptor for the No signal. However, this assumption was "

Later disproved after confirmation that ris is an HA receptor was obtained. Cells that co-express zto are unable to bind NI, which confirms that zto does not directly interact with NA (IMizze, (1996) Mayshge 384:119-120). Rather, the binding of Zopis peddepod (ZNN) to its receptor RTSN is believed to;" prevents the normal inhibition of the seven-helical transmembrane protein ztoiepei (ZMO) by the receptor

RTSN.

Recently, it was reported that the appearance of the second mutation occurs in sporadic basal cell carcinoma. (1998) Medicine 391:90-21), and primary neuroectodermal tumors of the central nervous system. (1998) Sapseg Kez 58:1798-803). ik The term "therapeutic index" refers to the therapeutic index of the drug, defined as LD5o/EDrvo. -And As used herein, the term "transformed cells" refers to cells that spontaneously enter a state of uncontrolled growth, that is, they acquire the ability to grow through an indefinite number of divisions in culture. o Transformed cells can be characterized by terms such as neoplastic, anaplastic, and/or 4) hyperplastic, which refers to the loss of control of their growth.

The term "acylamino" is known to those skilled in the art and refers to a group that can be represented by the general formula: o o . them) What and 60 . where K» is defined above, and K'4 represents hydrogen, alkyl, alkenyl or - (CH 25 )t-Ka, where t and Ka are as defined above.

The term "aliphatic group" refers to an aliphatic group of a straight, branched, or closed chain hydrocarbon and includes saturated and unsaturated aliphatic groups such as alkyl, alkenyl, and alkynyl.

The terms "alkenyl" and "alkynyl" refer to analogs of unsaturated aliphatic groups capable of being substituted by alkyls as described above, but which contain at least one double or triple bond, respectively.

The terms "alkyl" or "alkyl" as used herein refer to an alkyl group as defined above with an oxygen radical attached thereto. Typical alkyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, etc. groups "Ether" is two hydrocarbons covalently linked by oxygen. Accordingly, the alkyl substituent means that the alkyl of the ether is alkyl, which, for example, can be one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CHNo)t-Ka, where t and Ka are defined above.

The term "alkyl" refers to radicals of saturated aliphatic groups, including straight alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In a preferred embodiment, the straight or branched alkyl chain has 30 or fewer carbon atoms in its backbone (eg, C 4 -C 20 in a straight chain, C 3 -C 20 in a branched chain), and even more preferably 20 or less. Similarly, cycloalkyls are preferred if they have from 3 to 10 carbon atoms in their ring structure, and even more preferably if they have 5, 6 or 7 carbon atoms in their ring structure.

Furthermore, the term "alkyl" (or "lower alkyl"), as used throughout the description, examples, and claims, shall include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl groups, having substituents that replace hydrogen near one or more carbon atoms of the hydrocarbon skeleton. Such substituents may contain, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), 2-alkyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfohydryl, alkylthio, sulfate, sulfonate, sulfoamyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic groups. As should be understood by those skilled in the art, the groups that are substituents in the hydrocarbon chain may themselves, accordingly, be substituted. For example, substituted alkyl substituents may include substituted and unsubstituted amino, azido, imino, amido, phosphoryl (including phosphonate and thiophosphinate), sulfonyl (including sulfate, sulfonamido, sulfoamyl, and sulfonate), and silyl groups, as well as ethers, alkylthio groups, carbonyls (including ketones, aldehydes, carboxylates and ethers), -SEz, -CM and similar groups. and)

Examples of substituted alkyls are given below. Cycloalkyls may, in addition, be substituted by alkyls, alkenyls, alkoxy and alkylthio groups, aminoalkyls, carbonyl-substituted alkyls, -C3, -CM and similar groups. unless the number of carbon atoms is otherwise defined, the term "lower alkyl" as used herein means an alkyl group as defined above, but having from one to ten carbon atoms, preferably from one to six carbon atoms, in its skeletal structure . Similarly, "lower alkenyl" and "lower alkynyl" have similar M chain lengths. Throughout the application, alkyl groups are preferably lower alkyl. In preferred embodiments, the substituent designated herein as alkyl is lower alkyl. Me

The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur y radical attached to it. In preferred embodiments, the "alkylthio" group is represented by one of -5-alkyl, -5-alkenyl, -5-alkynyl, and -5-(CH0)t-Ka, where t and Ka are defined above. Examples of alkylthio groups include methylthio, ethylthio, and the like.

The terms "amine" and "amino" are well known to those skilled in the art and refer to both unsubstituted and substituted amines, such as, for example, a group that can be represented by the general formula: ra Ge y"? Ko chi shk ka

B, -I se) where each of Ko, Ko and Ko independently represent hydrogen, alkyl, alkenyl, "CH 2)t-Ka, or Kye and Ko, taken together with the M atom to which they are attached to form a heterocycle, having from 4 to 8 atoms in a ring structure; Ka represents aryl, cycloalkyl, cycloalkenyl, heterocycle or polycycle; and t is zero or an integer 1 in the range of 1 to 8. In preferred embodiments, only one of Co or Co can be a carbonyl, for example, Ka, and Co and the nitrogen together do not form an imide. In some such embodiments, neither K»e nor Co is a carbonyl attached to M, e.g., the amine is not an amide or an imide, and the amine is predominantly basic, e.g., its conjugate acid has a pKa value greater than 7. In even more preferred embodiments each of Co and Co (and possibly Co) independently represents hydrogen, alkyl, alkenyl, or -(CHNo)t-Ca. Thus, the term "alkylamine" as used herein means an amino group as defined above having a substituted or unsubstituted alkyl attached thereto, i.e. at least

HF) one of Ka and K:o is an alkyl group. 7 The term "amido" is commonly known as an amino-substituted carbonyl and contains a group that can be represented by the general formula: 60 Ho

Same in

where Co, Co is defined above. Preferred amide embodiments will not include imides, which can be unstable.

The term "aralkyl" as used herein refers to an alkyl group substituted with an aryl group (eg, an aromatic or heteroaromatic group).

The term "aryl" as used herein includes 5-, 6-, and 7-membered monocyclic aromatic groups that may contain from zero to four heteroatoms, e.g., benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl substituted groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring may be substituted in one or more positions of the /o ring by such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkyl, amine, nitro, sulfohydryl, imine, amide , phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ester, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclic, aromatic or heteroaromatic groups, -SEz, -CM or the like. 7/5 The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are shared by two adjacent rings (the rings are "fused rings"), wherein at least one of the rings is aromatic and other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or heterocycles.

The term "carbocycle" as used herein refers to an aromatic or non-aromatic ring in which each 20 ring atom is carbon.

The term "carbonyl" is well-known and includes such groups that can be represented by the general formula: о о с что "M min or -x-M vu o where X is a bond or represents oxygen or sulfur, and K 44 represents hydrogen, alkyl, alkenyl, -"CH 2)t-Ka, or c zo a pharmaceutically acceptable salt, K' 44 represents hydrogen, alkyl, alkenyl, -(CH o)t-Kv, where t and Ka are defined above. If X is oxygen and K.44 or K4 is not hydrogen, the formula represents an "ester". If X is oxygen and Ku is as defined above, the group is considered here as a carboxyl group, if K is hydrogen, the formula represents a "carboxylic acid". If X is oxygen and K'44 is hydrogen, the formula represents "formate". In general, if the oxygen atom of the above formula is replaced by sulfur, the formula represents a me) "thiocarbonyl" group. If X is sulfur and K/4 or KU is not hydrogen, the formula represents a "complex thioether". uh-

If X is sulfur and K.4 is hydrogen, the formula represents a "thiocarboxylic acid." If X is sulfur and K'44 is hydrogen, the formula represents "thioformate." On the other hand, if X is a bond and K 44 is not hydrogen, the above formula represents a "ketone" group. If X is a bond and K 44 is hydrogen, the above formula represents an "aldehyde" group. "

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. The heteroatoms are mainly boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The terms "heterocyclyl" or "heterocyclic group" refer to 3-membered to 10-membered rings;» structures, preferably from 3- to 7-membered rings, the ring structures of which include one to four heteroatoms.

Heterocycles can also be polycycles. Heterocyclic groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, -I pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolysine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cynoline, pteridine, carbazole, carboline, phenanthridine, se) acridine, pyrimidine, phenatroline, phenazine, phenarsazine, phenothiazine, furazane, phenoxazine, pyrrolidine, oxolane, -I thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, bultams, sultones and the like. The heterocyclic ring can be substituted in one or more positions with such substituents as described above, such as halogen, alkyl, aralkyl, alkenyl, 4) alkynyl, cycloalkyl, hydroxyl, amine, nitro, sulfohydryl, imine, amide, phosphate, phosphonate , phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic, aromatic or two Heteroaromatic groups, -SEz, -CM or the like.

As used herein, the term "nitro" means -MO 5; the term "halogen" means -P, -CI, -Bg or -I; term

F) "sulfhydryl" means -5H; the term "hydroxyl" means -OH; and the term "sulfonyl" means -505. ka "Phosphonamidite" can be represented by the general formula: 60 Kav c

And | ay 2 runes union. kyu uk Play ag o-, or og? Okemik e MK.) Ve b5 where K»o and K.o are as defined above, CO» represents O, 5 or M, and Kv represents lower alkyl or aryl.

"Phosphoramidite" can be represented by the general formula: и т -00238-0-- teaYut OK.

And OR

M(KaR Bo miv Ki where K»o and Co are as defined above, CO» represents O, 5 or M. 70 "Phosphoryl" can generally be represented by the formula: te and OK where OS represents 5 or O, and K 46 represents hydrogen, lower alkyl, or aryl. When, for example, alkyl is used as a substituent, the phosphoryl group of phosphorylalkyl can be represented by the general formula: hydrogen, lower alkyl or aryl, C 2 represents O, C or M. When C); is 5, then the phosphoryl group is "phosphorothioate".

The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or heterocyclyl) in which two or more carbon atoms are shared by two adjacent rings, e.g., the rings are "condensed rings". Rings in which non-adjacent atoms are common are called "town" rings. Each of the rings of the monocycle can be substituted with such substituents as described above, such as, for example, halogen, alkyl, aralkyl, alkenyl, -alkynyl, cycloalkyl, hydroxyl, amine, nitro, sulfohydryl, imine, amide, He») phosphate, phosphonate, phosphinate , carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic, aromatic or heteroaromatic groups, -SEz, -CM or the like.

The term "protecting group" as used herein refers to temporary substituents that protect a potentially reactive functional group from unwanted chemical transformations. Examples of such protecting groups include carboxylic esters, silyl esters of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of chemistry of protective groups is considered in the book Sgeepe, T. MU.; MuUtsiv, R. b. M. Rgoyesiime (zgoiMrz s ip Ogdapis bupipeziv, 279 ed.; UMieu: Mem/ Hogk, 19911. :z» The term "selenoalkyl" refers to an alkyl group having a substituted seleno group attached to it. Examples of "selenoesters" that may be alkyl substituted can be one of -Ze-alkyl, -Ze-alkenyl, -Ze-alkynyl, and -56e-(CHNa )t-Ka, where t and Ka are as defined above. - As used herein, the term "substituted" is intended to refer to all permissible substituents of organic compounds. Broadly speaking, permissible substituents include acyclic and cyclic, branched and and is) unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. - Exemplary substituents include, for example, those described above. Acceptable substituents may be one or more of the same or different for the relevant organic compounds. For the purposes of this invention, o heteroaromatics such as nitrogen may have hydrogen substituents and/or any acceptable substituents of the organic compounds described herein which correspond to the valence of the heteroatoms. The present invention does not provide for any restrictions regarding the permissible substitutes of organic compounds.

It is understood that the terms "substitution" or "substituted" carry an implicit stipulation that such substitution corresponds to the valence of the substituted atom and the substituent, and that the substitution results in a stable compound, such as one that does not undergo spontaneous transformations such as rearrangement, cyclization, elimination, and etc. (F) The term "sulfamoyl" is well-known and contains a group that can be represented by the general formula: име) д т» 60 ше Стш- Кк и о КЕ. where Ki and Co are as defined above. 65 The term "sulfate" is commonly known and contains a group that can be represented by the general formula:

it

II

-0-5-OK,1

HER o de KA is as defined above.

The term "sulfonamido" is well-known and contains a group that can be represented by the general formula:

Ka de K and K44 are as defined above.

The term "sulfonate" is commonly known and contains a group that can be represented by the general formula:

where C is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms "sulfoxide" or "sulfinyl" as used herein refer to a group that can be represented by the general formula:

THESE

- seams s y y s. and and and and where Ka44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl IV) or aryl.

Analogous substitutions can be made in alkenyl and alkynyl groups to give, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, He»)thioalkynyls, carbonyl-substituted alkenyls and alkynyls. As used herein, the definition of each term, eg, alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

The terms trifyl, tosyl, mesyl, and nonafyl are commonly known and refer to the trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are commonly known and refer to trifluoromethanesulfonate esters, p-toluenesulfonate esters, methanesulfonate c esters, and nonafluorobutanesulfonate ester functional groups and molecules that contain these groups, respectively.

The abbreviations Me, Ex, RY, Ti, M, Tv, Me represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl, and methanesulfonyl, respectively. A more complete list of abbreviations, 15 commonly used by organic chemists, is published in the first edition of each volume ("Journal

of Organic Chemistry" (otsgpa! oi Ogdapis Spetivigu)!; this list is usually presented in the form of a table under the name (Se) Standard List of Abbreviations. Abbreviations contained in this List and all abbreviations that are commonly used by organic chemists , are used here with appropriate references.

Certain compounds of the present invention may exist in specific geometric or stereoisomeric forms. According to the proposed invention, all such compounds, including cis- and trans-isomers, c» K- and B-enantiomers, diastereomers, (0) - isomers, (Y) - isomers, their racemic mixtures and other mixtures, are considered as being within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in the present invention. 59 If, for example, the corresponding enantiomer of a compound of the invention is desired, it can be obtained

HF) by asymmetric synthesis or derivatization with a chiral auxiliary group, with the resulting 7 diastereomeric mixture separated and the auxiliary group cleaved to ensure purity of the desired enantiomer. Alternatively, if the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts may be formed with the appropriate optically active acid or base, followed by resolution of the diastereomers and appropriate formation by fractional crystallization or by known chromatographic means and subsequent reduction of pure enantiomers.

Proposed equivalents of the above compounds contain similar compounds that have the same general properties (eg, the ability to inhibit Ppeddepod signaling), with one or more simple substitution variations made that do not adversely affect the compound's efficacy. In general, the compounds of the present invention may be prepared by methods illustrated in general reaction schemes, such as those described below, or modifications thereof, using readily available starting materials, reagents, and conventional synthetic procedures. In these reactions, it is also possible to use variants that are known but not mentioned here.

For the purposes of this invention, chemical elements are identified in accordance with the Periodic Table of Elements, version SA, (Textbook of Chemistry and Physics (Napadbook oi Spetivigu apa RNuvisv, 67!" Ei., 1986-87)). Also for the purposes of this invention, the term " "hydrocarbon" is considered to include all permissible compounds having at least one hydrogen atom and one carbon atom. Broadly speaking, permissible hydrocarbons include non-cyclic and 70 cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds, which may be substituted or unsubstituted.

IP Examples of compounds according to the invention

As described in more detail below, it is understood that the methods of the invention can be implemented using many different small molecules that can be easily identified, for example, by a drug screening method as described herein. For example, the compounds used in the methods according to the invention can be represented by the general formula (1): ge A ad: po) U

TA school in o

Formula I so de, as far as valency and stability allow, yu

X and 7 are independently -М(К7)-, -О-, -5-, -(К7)М-МЩ(К7)-, - ОМ(К7)- or a simple bond, preferably -М(К7) -, -O-, -5- or a simple bond; -

M is -F(50)-, -C(55)-, -C(-MA)-, ZO" or 5O, preferably -C(-0)-, ZO" or -C(-5)-; Gee!

And there is O, 5 or MK, better O or MN, and best of all MN;

C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring, condensed with the ring to which it is attached, preferably an aryl or heteroaryl ring;

Ar is a substituted or unsubstituted aryl or heteroaryl ring, such as a substituted or unsubstituted phenyl ring; "

Ku represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or - 70 cycloalkyl, including polycyclic groups; c Co represents from 0 to 4 substituents on the ring to which it is attached, such as halogen, lower alkyl, c" lower alkenyl, aryl, heteroaryl, carbonyl (eg, ester, carboxyl or formyl), thiocarbonyl (eg , complex thioether, thiocarboxylate or thioformate), ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, 9U-Ka, 9U -OH, O-lower alkyl, dU-lower alkenyl, .-Ka, У-ЗН, 9У-МН»о, forms of the above are also protected, or any two Ко when they occur more than once in a cyclic or ( Ce) polycyclic structure, can be taken together with the formation of 4-8-membered cycloalkyl, aryl or heteroaryl;

KE, in each case independently, is H, lower alkyl (e.g., substituted or unsubstituted), --cycloalkyl 7 (e.g., substituted or unsubstituted), y9-heterocyclyl (e.g., substituted or unsubstituted), -aryl cl 20 (e.g., substituted or unsubstituted), --heteroaryl (eg, substituted or unsubstituted);

Ka is, in each case, independently H, lower alkyl (eg, substituted or unsubstituted), cycloalkyl c" (eg, substituted or unsubstituted), heterocyclyl (eg, substituted or unsubstituted), aryl (eg, substituted or unsubstituted), or heteroaryl (eg substituted or unsubstituted); and

Y, in each case independently, is a chain containing from 0 to 8 (preferably 0 to 4) units, 29 selected from SK", MK, O and 5, where K, in each case independently, is H or lower alkyl

GF) In certain embodiments, at least one of 7 and X is not a simple bond. In some embodiments, X-U-7 includes an amide, a urea, or a sulfonamide. In some embodiments, X is selected from -M(K8)-, -O-, -5-, and preferably is MH.

In certain embodiments, K 4 includes an aryl or heteroaryl ring, optionally substituted with 1 to 5 substituents such as nitro, halogen, cyano, lower alkyl, acylamino (eg, K 8d -C(-O)MH-), alkoxy, 60 alkylamino, substituted or unsubstituted cycloalkyl, heterocyclyl, aryl or heteroaryl fused to an aryl or heteroaryl ring.

In some embodiments, X and the ring containing A are arranged on Ag in a meta (ie, 1,3) relationship.

In certain embodiments, c is a phenyl or piperidine ring.

In certain embodiments, U is absent. for In certain embodiments, Co is from 1 to 4 substituents selected from halogen, cyano, nitro,

alkoxy, amino, acylamino (eg, Ca-C(-O)MH-), substituted or unsubstituted cycloalkyl, heterocyclyl, aryl or heteroaryl co-fused with 0, and substituted or unsubstituted lower alkyl.

In certain embodiments, the compound of the invention is selected from the compounds depicted in Figure 32.

In some embodiments, the compounds of the present invention may be represented by the general formula (I):

And pe-y ia

Or, shk

And who

Formula I where, as far as valency and stability allow,

X and 7 are independently -М(К7)-, -О-, -5-, -(К7)М-МЩ(К7)-, - ОМ(К7)- or a simple bond, preferably -М(К7) -, -О-, -5- сч or a simple connection; Gee)

M is -F(50)-, -C(55)-, -"C(-MA)", 5O" or 5O, preferably -C(-0)-, ZO" or -C(-5)-;

And there is O, 5 or MK, better O or MN, and best of all MN;

C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring fused with the ring to which it is attached, preferably an aryl or heteroaryl ring; co 3 o Ku represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or IS o) cycloalkyl, including polycyclic groups;

Co represents from 0 to 4 substituents on the ring to which it is attached, such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl (e.g., ester, carboxyl, or formyl), F) thiocarbonyl (e.g. , complex thioether, thiocarboxylate or thioformate), ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, - phosphoryl, phosphonate, phosphinate, 9U-Ka, 9U-OH, O-lower alkyl, dU-lower alkenyl, .-Ka, U-ZN, 9U-MH»o, protected forms of the above, or any two Co when they occur more than once in a cyclic or polycyclic structures, can be taken together with the formation of 4-8-membered cycloalkyl, aryl or heteroaryl. " du Cz represents from 0 to 4 substituents on the ring to which it is attached, such as halogen, hydroxyl, c alkoxy, amino, acylamino, cyano, nitro, substituted or unsubstituted lower alkyl and acyl, preferably halogen, c lower alkoxy , or substituted or unsubstituted lower alkyl; :z» KE, in each case independently, is H, lower alkyl (eg, substituted or unsubstituted), --cycloalkyl (eg, substituted or unsubstituted), y9-heterocyclyl (eg, substituted or unsubstituted), -aryl (eg, substituted or unsubstituted), --heteroaryl (eg, substituted or unsubstituted); - 15 Ka, in each case independently, is H, lower alkyl (eg, substituted or unsubstituted), cycloalkyl (eg, substituted or unsubstituted), heterocyclyl (eg, substituted or unsubstituted), aryl (Ce) (eg, substituted or unsubstituted ) or heteroaryl (eg, substituted or unsubstituted); and - Y, in each case independently, is a chain containing from 0 to 8 (preferably from 0 to 4) units selected from the group consisting of SK", MK, O and 5, where K, in each case independently , is H or lower alkyl. 1 50 In certain embodiments, at least one of 7 and X is not a simple bond. In some embodiments, X-U-7 includes an amide, urea, or sulfonamide. In some embodiments, X is selected from the group consisting of -M(K8)-, -O-, -5-, and preferably is MH.

In certain embodiments, K 4 includes an aryl or heteroaryl ring, optionally substituted with 1 to 5 substituents such as nitro, halogen, cyano, lower alkyl, acylamino (eg, K 8 -C(-O)MH-), alkoxy, 59 alkylamino, substituted or unsubstituted cycloalkyl, heterocyclyl, aryl or heteroaryl, which are fused with

HF) aryl or heteroaryl ring. 7 In certain embodiments, c is a phenyl or piperidine ring.

In certain embodiments, the .// is absent. In certain embodiments, Co is 1 to 4 substituents selected from halogen, cyano, nitro, alkoxy, amino, acylamino (eg, Ca-C(-O)MH-), substituted or unsubstituted cycloalkyl, heterocyclyl, aryl or heteroaryl co-fused with 0 and substituted or unsubstituted lower alkyl.

In some embodiments, K c includes a substituent, such as substituted or unsubstituted alkyl or halogen, in the para-position to both X and the ring containing A.

In certain embodiments, the compound of the invention is selected from the compounds depicted in Figure 32. 65 As described in more detail below, it is understood that the methods of the invention can be implemented using many different small molecules that can be easily identified, for example, by a drug screening method as described herein. For example, the compounds used in the methods according to the invention can be represented by the general formula (FI): er 0 and -k

Shk - I

Formula Ш where, as far as valency and stability allow,

X and 7 are independently -М(К7)-, -О-, -5-, -(К7)М-МЩ(К7)-, - ОМ(К7)- or a simple bond, preferably -М(К7) -, -O-, -5- or a simple bond;

M is -F(50)-, -C(55)-, -C(-MA)-, ZO" or 5O, preferably -C(-0)-, ZO" or -C(-5)-;

And there is O, 5 or MK, better O or MN, and best of all MN;

C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring fused with the ring to which it is attached, preferably an aryl or heteroaryl ring; with

O is absent or is SK", MK, O and 5, where K, in each case independently, is H or lower alkyl; at

Ar is a substituted or unsubstituted aryl or heteroaryl ring, such as a substituted or unsubstituted phenyl ring;

Ku represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or cycloalkyl, including polycyclic groups; at

Co is from 0 to 4 substituents on the ring to which it is attached, such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl (eg, ester, carboxyl or formyl), thiocarbonyl (eg, thioether, thiocarboxylate or thioformate), ketone, aldehyde, amino, acylamino, etc. amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, Ge! phosphoryl, phosphonate, phosphinate, 9U-Ka, 9U-OH, O-lower alkyl, dU-lower alkenyl, .-Ka, U-ZN, 9U-MH»o, protected 32 forms of the above, or any two Co, when they occur more than once in a cyclic or polycyclic structure, can be taken together to form a 4-8 membered cycloalkyl, aryl or heteroaryl;

C7, in each case independently, is H, lower alkyl (eg, substituted or unsubstituted), --cycloalkyl (eg, substituted or unsubstituted), y9-heterocyclyl (eg, substituted or unsubstituted), -aryl" (eg, substituted or unsubstituted), --heteroaryl (eg, substituted or unsubstituted);

Ka is, in each case, independently H, lower alkyl (e.g., substituted or unsubstituted), cycloalkyl C3 (e.g., substituted or unsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl"" (e.g., substituted or unsubstituted) or heteroaryl (eg, substituted or unsubstituted); and "Y, in each case independently, is a chain containing from 0 to 8 (preferably 0 to 4) units selected from SK", MK, O and 5, wherein K, in each case independently, is H or lower alkyl.

In certain embodiments, at least one of 7 and X is not a simple bond. In some embodiments, X-U-7 includes an amide, a urea, or a sulfonamide. In certain embodiments, X is selected from -M(K8)-, -O-, -5-, and preferably is MH.

Ge) In certain embodiments, K 4 includes an aryl or heteroaryl ring, optionally substituted with 1 to 5 substituents such as nitro, halogen, cyano, lower alkyl, acylamino (eg, K 8-C(-O)MH-), alkoxy, C-alkylamino, substituted or unsubstituted cycloalkyl, heterocyclyl, aryl or heteroaryl, which are fused to a C 20 aryl or heteroaryl ring.

In some embodiments, X and the ring containing A are arranged on Ag in a meta (ie, 1,3) relationship. c» In certain embodiments, c is a phenyl or piperidine ring.

In certain embodiments, the .// is absent.

In certain embodiments, Co is from 1 to 4 substituents selected from halogen, cyano, nitro, 20 alkoxy, amino, acylamino (eg, Ca-C(-O)MH-), substituted or unsubstituted cycloalkyl, heterocyclyl,

HF) aryl or heteroaryl, co-condensed with b, and substituted or unsubstituted lower alkyl. In certain embodiments, the compound of the invention is selected from the compounds shown in Fig.32. o In certain embodiments, the compounds used in the methods according to the invention can be represented by the general formula (IM): 60 b5

Kz

J

Zv

M x-th x

And in

The TU formula where, as far as valency and stability allow,

X and 7 are independently -М(К7)-, -О-, -5-, -(К7)М-МЩ(К7)-, - ОМ(К7)- or a simple bond, preferably -М(К7) -, -O-, -5- or a simple bond;

M is -F(50)-, -C(55)-, -C(-MA)-, ZO" or 5O, preferably -C(-0)-, ZO" or -C(-5)-;

And there is O, 5 or MK, better O or MN, and best of all MN;

C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring fused with the ring to which it is attached, preferably an aryl or heteroaryl ring;

O is absent or is SK", MK, O and 5, where K, in each case independently, is H or lower alkyl; with

K; is H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or o-cycloalkyl, including polycyclic groups;

Co is from 0 to 4 substituents on the ring to which it is attached, such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl (eg, ester, carboxyl, or formyl), thiocarbonyl (eg, thioether ) 9U-OH, O-lower alkyl, dU-lower alkenyl, .-Ka, U-ZN, 9U-MH»o, protected forms of the above, or any two Co when they occur more than once in a cyclic or - polycyclic structure, can be taken together with the formation of 4-8-membered cycloalkyl, aryl or heteroaryl. (o) z represents from 0 to 4 substituents on the ring to which it is attached, such as halogen, hydroxyl, m alkoxy, amino, acylamino, cyano, nitro, substituted or unsubstituted lower alkyl and acyl, preferably halogen, lower alkoxy , or substituted or unsubstituted lower alkyl;

KE, in each case independently, is H, lower alkyl (eg, substituted or unsubstituted), --cycloalkyl (eg, substituted or unsubstituted), y9-heterocyclyl (eg, substituted or unsubstituted), -aryl "di (eg, substituted or unsubstituted), --heteroaryl (eg, substituted or unsubstituted); -at

Ka is, in each case, independently H, lower alkyl (e.g., substituted or unsubstituted), cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl :c" (e.g., substituted or unsubstituted) whether heteroaryl (eg, substituted or unsubstituted); and

Y, in each case independently, is a chain containing from 0 to 8 (preferably 0 to 4) units selected from the group consisting of SK", MK, O and 5, where K, in each case independently, is H or lower alkyl. - 75 In certain embodiments, at least one of 7 and X is not a simple bond. In some embodiments, X-U-7 includes an amide, urea, or sulfonamide. In some embodiments, X is selected from the group consisting of -M(K8)-, -O-, -5-, and preferably (Ce) is MH. - In certain embodiments, K 4 includes an aryl or heteroaryl ring, optionally substituted with 1 to 5 substituents, such as nitro, halogen, cyano, lower alkyl, acylamino (eg, K 8-C(-O)MH-), alkoxy , 1 50 alkylamino, substituted or unsubstituted cycloalkyl, heterocyclyl, aryl or heteroaryl, which are fused to a c» aryl or heteroaryl ring.

In certain embodiments, c is a phenyl or piperidine ring.

In certain embodiments, the .// is absent.

In certain embodiments, Co is from 1 to 4 substituents selected from halogen, cyano, nitro, alkoxy, amino, acylamino (eg, Ca-C(-O)MH-), substituted or unsubstituted cycloalkyl, heterocyclyl,

HF) aryl or heteroaryl, cofused with 0, and substituted or unsubstituted lower alkyl. 7 In some embodiments, K z includes a substituent, such as substituted or unsubstituted alkyl or halogen, in the para-position to both X and the ring containing A. In certain embodiments, the compound of the invention is selected from the compounds shown in FIG. 32. for According to some of the embodiments, the proposed antagonists can be selected based on their selectivity to peddepods of the signaling pathway. This can be selectivity in relation to the peddenpod pathway compared to other pathways, or selectivity between specific peddeniod pathways, for example, ris-1, ris-2, etc.

In some preferred embodiments, the provided inhibitors inhibit signal transduction mediated by loss of function of ris, gain of function of peddepod, or gain of function of stooiyepei at an EO of 50 that is 1 mM or less, preferably 1 µM or less, and even more preferably 1 mM or less. Similarly, in certain preferred embodiments,

the proposed inhibitors inhibit the activity of the peddeiod pathway with an inhibition constant of K; less than 1 TOnM, preferably less than 1 nM, even more preferably less than 0.1 nM.

In some embodiments, a small molecule is chosen for use because it is more selective for one isoform of raisin compared to another, for example, 10-fold, and more preferably at least 100 or even 1000-fold more selective for a single raisin isoform. path (fig-1, fig-2) in comparison with the other.

In some embodiments, a compound that is an antagonist of the peddeiod pathway is selected so that it selectively antagonizes peddepod activity over protein kinases other than protein kinase A 7/0 "PKA", such as protein kinase C (PKS); for example, the compound modulates the activity of the peddepod pathway by at least an order of magnitude more than it modulates the activity of another protein kinase, preferably at least two orders of magnitude more, more preferably at least three orders of magnitude more. So, for example, the best inhibitor of the peddepod pathway can inhibit peddepod activity with K at least an order of magnitude lower than K; inhibition of PKS, preferably at least two orders of magnitude lower, even better at least three orders of magnitude /5 Magnitude lower. In certain embodiments, K; for inhibition of PKA less than 1 ONM, better less than 1 NM, even better less than 0.1 nM.

THEM. Examples of application of the method and compositions

Another aspect of the present invention relates to the modulation of the differentiated state, survival and/or proliferation of cells with loss of function, loss of function, or gain of function by contacting the cells with a peddepod antagonist according to the method provided and taking into account the circumstances present. .

For example, this invention envisages that in the light of scientific discoveries regarding the possibility of wide participation of ris, peddenode and ztoipeped in the formation of an ordered spatial arrangement of differentiated tissues in vertebrates, the proposed method can be used as part of the process of production and/or preservation of a large number of diverse tissues in vertebrates - both ip mygo and ip mimo. Antagonist

Peddepod, both induced and anti-induced in relation to the proliferation or differentiation of a certain i) tissue, can be, if desired, any of the preparations described above.

For example, the proposed method can be used in methods related to tissue cultures, in which, for both genetic and biochemical reasons, phenotypes with ris - loss of function, c zo Ppeddenod - gain of function and ztooiped - acquisition of function can occur. functions. Research has shown that neuronal culture systems are fundamental and necessary tools for the study of neural development, as is the identification of neurotrophic factors such as nerve growth factor (GNF), ciliary M neurotrophic factor (MNF), and brain-derived neurotrophic factor (MNF). . One of the options for using the proposed method can be its application in cultures of stem neuronal cells, as well as in the use of such cultures for the production of new neurons and glia. In such embodiments of this method, cultured cells may be contacted with a peddepod antagonist of the invention to alter the rate of proliferation of neuronal stem cells in culture and/or alter the rate of differentiation, or preserve the integrity of the culture of certain terminally differentiated neuronal cells. In one of the examples of implementation, the proposed method can be applied, for example, to the culture of sensory neurons, or alternatively, motoneurons. Such neuronal cultures can be used as conventional test systems and, in addition, as sources of cells for cell implantation in therapeutic treatment. . According to the proposed invention, a large number of progenitor neural cells, which are not form tumors, can be continuously maintained, and their rate of proliferation and differentiation can be influenced by contact with peddepod antagonists according to the present invention. In general, the proposed method includes the steps of isolating neural progenitor cells from an animal, maintaining these cells as ip myo or ip mimo, preferably in the presence of growth factors, and regulating the differentiation of these cells into specific neural phenotypes, such as neurons and glia , by contacting these cells with the antagonist peddenpsod. Progenitor cells are under the influence of a tone inhibitor, which maintains the progenitors in a suppressed state until there is no need for their differentiation. However, recently methods have been proposed that ensure the proliferation of these cells, and unlike neurons, which are terminally differentiated and therefore no longer divide, these cells can be obtained in unlimited quantities and are very 4) well suited for transplantation in heterologous and autologous recipient organisms with neurodegenerative diseases.

By "progenitors" we mean stem cells that are oligopotent or multipotent, capable of dividing without restriction, and under certain conditions can produce daughter cells that are terminally differentiated, such as neurons and glia. These cells can be used for transplantation in heterologous

F) or an autologous recipient organism. Heterologous is considered to be an organism of the recipient, other than the animal from which the progenitor cells were taken. The same organism from which the cells were taken is considered autologous.

Cells can be obtained from the nervous tissues of embryos, newborns, young and adults of 60 any animals. All multicellular animals that have nervous tissue are considered any animals.

More specifically, any fish, reptile, bird, amphibian, mammal, and the like are meant.

The best donors are mammals, especially mice and humans.

In cases where a heterologous donor animal is used, it is euthanized, and the brain and specific parts of interest are separated under sterile conditions. Areas of special interest in the brain include any sites from which progenitor cells can be harvested to be used to restore the function of a degenerating area of the recipient's brain. These parts contain areas of the central nervous system (CNS), including the cerebral cortex, cerebellum, midbrain, brainstem, spinal cord, and ventricular tissue; as well as areas of the peripheral nervous system (PNS), carotid body, and adrenal medulla. More specifically, these areas include the sites where the basal ganglia are located, and best of all, the striatum, which consists of the caudate and lenticular nuclei, or various cell groups such as the lenticular nucleus pallidus, subthalamic nuclei, basal nuclei that degenerate, such as established, in patients with Alzheimer's disease, or the substantia nigra of the midbrain, which degenerates, as established in patients with Parkinson's disease.

Heterologous human progenitor neural cells can be obtained from fetal tissues obtained from 7/0 elective abortion or from newborn, juvenile and adult donors. Autologous nerve tissue can be obtained by biopsy, or during neurosurgical operations where nerve tissue is removed, particularly in operations on patients with epilepsy, and especially during temporal lobectomies or hippocampotomies.

Cells can be obtained from donor tissues by detaching individual cells from the extracellular tissue matrix. Dissociation can be carried out by any known procedure, for example, 7/5 treatment with enzymes such as trypsin, collagenase and the like; or using a mechanical method of detachment with a blunt instrument or carefully with a scalpel - so that the specific types of cells removed from this tissue can continue to develop. Separation of fetal cells from the matrix can be carried out in a tissue culture medium, although artificial cerebrospinal fluid (CSF) is the best medium for separating young or adult cells. Normal SHSMR contains 124mM Mass, 5mM KSI, 1.3mM Masi, 2mM Sasi, 26mM Manso»z and 10mM D-glucose. Low-Ca artificial CMR contains the same ingredients, except that the concentration of CaCl is 3.2 mM and the concentration of CaCl is 0.1 mM.

The isolated cells can be transferred to any known cell culture medium capable of supporting cell growth, including MEM, DMEM, KRMI and E-12 and the like, with appropriate additional substances required for cell metabolism, such as glutamine and other amino acids, vitamins, Ha

Minerals and useful proteins such as transferrin and the like, cell culture media capable of supporting cell growth, including MEM, DMEM, KRMI and E-12 and the like, with appropriate additional substances required for cell metabolism , such as glutamine and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like. The medium may also contain antibiotics to prevent contamination with yeast, bacteria, fungi, such as penicillin, streptomycin, gentamicin and the like. In some cases, the medium may contain serum obtained from cows, horses, chickens and other animals. Mixtures of DMEM and B-12 have the greatest advantages. at

Conditions for cultivation should be close to physiological ones. The pH value of the culture should be closer to the normal pH value in the body, it is desirable that the pH value be between 6 and 8, it is better if the pH is close to 7 and even better when the pH is about 7.4. Cells should be cultivated in temperature conditions close to physiological ones, it is good at 3020-402C, better at 3220-382C, and even better at 3520-3790. -

Cells can be grown in suspension or on solid substrates, but proliferation of progenitors is best in suspension, where large numbers of cells can be grown by forming "neurospheres" | see, for example, Keupoiaz ei ai (1992), Zsiepse 255; 1070-1709; and PCT publications U/093/01275, MU094/09119, MuO94/10292 " and MU/094/16718). In the case of stratification (or separation) of suspended cells, the flask should be shaken well, and the neurospheres will be able to settle to the bottom of the flask. Then the spheres are transferred to the BOml centrifuge tube and centrifuged at low speed. The medium is aspirated, the cells are resuspended in a small amount of medium with a growth factor, the cells are separated mechanically and resuspended in separate parts of the medium. ,» Cell suspensions in the culture medium are supplemented with any growth factor that ensures the proliferation of progenitor cells, and are "seeded" into any reservoir capable of holding cells, preferably culture flasks or closed bottles. Cells usually proliferate within 3-4 days in - I incubator at 372C, and proliferation can be repeated at any time thereafter by repeated cell separation and resuspension in fresh medium with growth factor.

In the absence of a substrate, cells rise from the bottom of the flask and continue to proliferate in suspension, creating a hollow sphere of undifferentiated cells. After approximately 3-10 days of incubation, proliferated sl 50 clusters (neurospheres) are fed every 2-7 days, or even better every 2-4 days by gentle centrifugation and resuspension in medium containing growth factor. After 6-7 days of incubation, individual cells in the neurospheres can be separated by physical separation from the neurospheres with a blunt instrument, preferably by trituration with a pipette.

Individual cells from the separated neurosphere are suspended in a culture medium containing a growth factor, and cell differentiation in culture can be controlled by adding (or resuspending) cells in the presence of a Peddepod antagonist.

To further illustrate other applications of the proposed Ppeddepod antagonists, it should be noted that intracerebral cell transplantation is an additional approach to the treatment of diseases of the central nervous system. For example, one of the possibilities to restore the function of damaged brain tissues involves the transplantation of such cells, extracted from the fetus or newborn animals, into the brain of an adult animal. and Rgetspa ei a/(1985) OMEitsgozsi 5:603-616)i. Fetal neurons from different parts of the brain can be successfully transplanted into the brain of an adult, and this significantly reduces the severity of behavioral disorders. For example, movement disorders due to dopaminergic lesions in the basal ganglia can be prevented by transplantation of embryonic dopaminergic neurons. Complex cognitive functions that have been impaired by damage to the neocortex can also be partially restored by transplantation of embryonic cortical cells. The proposed method can be used to regulate growth in culture, or, in the case of fetal tissues, especially neural stem cells, can be used to regulate the rate of differentiation of stem cells.

Stem cells used in accordance with the proposed invention are generally known. For example, several types of neural crest cells have been identified, some of which are multipotent and most likely represent uncommitted neural crest cells, and others that can generate only one cell type, such as sensory neurons, and which probably , represent committed progenitor cells. Role

The peddeiod of the antagonists, which they play in the proposed method, is manifested in the regulation of the differentiation of uncommitted progenitors or in the regulation of the further restriction of the developmental pathways of the 7/0 Committed progenitor cell before its transformation into a terminally differentiated neural cell.

For example, the proposed method can be used to regulate the differentiation of neural crest cells into glial cells, Schwann cells, chromaffin cells, cholinergic sympathetic or parasympathetic neurons, and peptidergic and serotonergic neurons. Neddeiodine antagonists can be used alone or in combination with other neurotrophic factors to enhance one or another pathway 7/5 differentiation of neuronal progenitor cells.

In addition to the implantation of cells cultured in the presence of the proposed peddepod antagonists, another aspect of the present invention relates to the therapeutic use of the peddepod antagonist to regulate the growth of neurons and other neuronal cells of both the central and peripheral nervous systems.

The ability of ris, peddeiod and stooieped to regulate neural differentiation during the development of the nervous system and possibly also in the mature state shows that under certain circumstances the Neddeipod antagonists proposed can be expected to influence mature neurons with respect to the preservation, function and aging of normal cells ; restoration and regeneration processes in chemically or mechanically damaged cells; therapy of degeneration in certain pathological conditions. In light of this understanding of the present invention, it is particularly recommended to use the proposed method in a treatment protocol for (prevention and/or amelioration of) neurological conditions resulting from: (i) acute, subacute or o chronic damage to the nervous system, including trauma, chemical damage, vascular damage and insufficiency (such as ischemia after a stroke), along with injuries of infectious/inflammatory and tumor origin; (her) aging of the nervous system, including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis (ALS) and similar diseases, as well as spinocerebral degenerations; and (im) chronic immunological diseases of the nervous system, including multiple sclerosis. i am

If necessary, the proposed method can also be used in the generation of nerve prostheses. M to restore the damaged function of the central and peripheral nervous systems. In particular, in cases where a flattened or severely damaged nerve ending is covered by a prosthetic device such as a tube around Me

From the axon, Peddepod antagonists can be added to this prosthetic device in order to regulate the rate of growth and regeneration of dendritic processes. Examples of nerve conduction channels are shown in US Patents 5,092,871 and 4,955,8921.

In another embodiment, this method can be used in the treatment of neoplastic or hyperplastic transformations occurring in the central nervous system. For example, a peddeipod antagonist can force such transformed cells to become postmitotic or apoptotic. The proposed method can be part of the treatment for, for example, malignant gliomas, meningiomas, medulloblastomas, neuroectodermal tumors and ependymomas. ;" In the best embodiment, the proposed method can also be used as part of the treatment process for malignant medulloblastoma and other primitive malignant neuroectodermal tumors of the CNS.

In another embodiment, the proposed method can be included in the treatment program for medulloblastoma. -I Medulloblastoma, a primitive neuroectodermal tumor, is the most common type of brain tumor in children.

Medulloblastoma is a primitive neuroectodermal tumor (PNEP) that grows in the posterior fossa. It accounts for approximately 25,905 of all pediatric brain tumors (MiPeg). Histologically, these are small cell tumors in the shape of a circle, usually they turn into real rosettes (a cell cluster in the shape of a rose), but they can sometimes differentiate into astrocytes, ependymal cells or neurons (Kogke; Kivipcez). PNEPs can grow in other parts of the brain, including the pineal gland (pineoblastoma) and the brain. Those of them, 4) which grow in the supratentorial part, are usually worse than those located in the opposite areas of the posterior cranial fossa.

Medulloblastoma/PNEPI are known for the fact that they can reappear after removal and can even metastasize to the bones. Therefore, preparation for treatment should include examination of the spinal cord to determine the presence of "distant metastases." Magnetic resonance imaging (MRI) with gadolinium almost

F) has completely replaced myelography for this purpose, and cerebrospinal fluid (CSF) cytology, which is performed after surgery, is a common procedure.

In other embodiments, the proposed method is used as part of a treatment program for ependymomas. Ependymomas account for about 1095 of all brain tumors in children. In most cases, ependymomas arise from ependyma of the ventricles of the brain and have the shape of rosettes, channels and perivascular rosettes under the microscope. In the clinical and histological obstetric protocols (CGAP) of examinations of 51 children with detected ependymomas, 3/4 of the tumors were histologically benign. Approximately 2/3 of them grew from the area of the 4th ventricle. 1/3 was in the supratentorial part. Data from the Program for Surveillance, Epidemiology, and Long-Term Evaluation of 65 Treatment Outcomes of the US National Cancer Institute (NCI), as well as data from the CGAP, indicate that tumors were most common in children under 4 years of age. The average age was 5 years. Due to the fact that many children with this disease were infants, they often needed combined treatment.

Another aspect of the proposed invention is that ris, peddenode and/or peddenode are included in the morphogenic signals involved in the formation of other organogenic pathways in vertebrates, in addition to neuronal differentiation as described above, playing an undeniable role in the formation of other endodermal patterns, as well as in mesodermal and endodermal processes of differentiation. Thus, in accordance with the proposed invention, compounds that contain peddeipod antagonists can be used both in cell cultures and in therapeutic methods involving the production and maintenance of non-neuronal tissue. 70 In one of the embodiments, the proposed method makes it possible to use the discovery of that property of ris,

Peddenode and/or ztooipeped, that they participate in controlling the development of stem cells, which are responsible for the formation of the digestive system, liver, lungs, and other organs derived from the primitive intestine. The ZAY performs the function of transmitting an inductive signal from the endoderm to the mesoderm, which is extremely important for the morphogenesis of the intestine. Thus, for example, pedopiod antagonists in the proposed method can be involved in the regulation of the development and maintenance of an artificial liver that can perform a large number of metabolic functions in the same way as a normal liver. In an exemplary embodiment, the proposed method can be used to regulate the proliferation and differentiation of alimentary canal stem cells to generate hepatocyte cultures that can be recruited to increase the amount of extracellular matrices or encapsulated in biocompatible polymers to create implantable artificial livers or use 2o extracorporeally.

In another embodiment, therapeutic compounds with Ppeddepod antagonists can be used in conjunction with the transplantation of such artificial livers, as well as embryonic liver structures, to regulate the process of acceptance of intraperitoneal implantation, vascularization, as well as ip mimo differentiation and maintenance of the implanted liver tissue. high school

In yet another embodiment, the proposed method can be used therapeutically to regulate the functions of the specified organs after physical, chemical or pathological damage. For example, therapeutic compounds that i) contain peddepod antagonists can be used as a temporary replacement for liver tissue repair in partial hepatectomy.

Formation of the pancreas and small intestine from the embryonic foregut depends on intercellular signaling between intestinal endodermal and mesodermal cells. In particular, it is believed that the differentiation of intestinal mesoderm into smooth muscle depends on signals from adjacent endodermal o cells. Zopis M is one of the possible mediators of endodermal signal transmission in the embryonic hindgut

Peddepod. |See, for example, Areidmivi ei aii. (1997) Sy. Vio! 7:801-4th4. The PP gene is expressed along the entire embryonic endoderm of the intestine, with the exception of the endoderm of the pancreas bud, which instead of Mezv expresses high levels of the homeodomain protein Irp/Rakhi! (ipzship rgotoieg Tasiog 1 - a transcription factor that regulates the development of the pancreas, / pancreatic homeobox gene), the main regulator of the early development of the pancreas. |AreiYdmivyi ei aiYi., see above), investigated whether the differential expression of ZAI in the primary intestine really controls the differentiation of the surrounding mesoderm into specialized mesodermal derivatives of the small intestine and pancreas. To test this, they used the promoter «|rP/RaXI! of the gene for selective expression of ZPI in the developing pancreatic epithelium. In Irp/Rakh1-5pi z c transgenic mice, the pancreatic mesoderm developed rather into smooth muscle and interstitial cells. Keigel (Sa|a), which are characteristic of the intestines, than in the pancreatic mesenchyme and spleen. Also, huh?" pancreatic explants under the influence of ZNAI undergo the same path of intestinal differentiation. These results demonstrate that differential expression of the endodermal derivative of ZPP controls the segmentation and differentiation of the surrounding mesoderm in different parts of the digestive tube. -I In the context of this invention, it is contemplated that the proposed peddeipod antagonists can be used to control or regulate the proliferation and/or differentiation of pancreatic tissue, both se) ip mimo and ip migo. -I There is a wide variety of conditions of proliferation and differentiation of pathological cells in which inhibitors

According to the proposed invention, certain therapeutic advantages can be provided, while the general strategy consists, for example, in the correction of aberrant insulin expression, or the modulation of differentiation. If we take 4) more broadly, the present invention relates to a method of inducing and/or maintaining a differentiated state, enhancing survival and/or proliferation of pancreatic cells by contacting the cells with the proposed inhibitors. For example, according to the invention, it is envisaged that in light of the established participation of ris, peddenpos and ov Ztooiyepei in the formation of a given spatial arrangement of pancreatic tissues, the proposed method can be part of the method of formation and/or maintenance of such tissue as ip myo and ip mimo. For example,

F) modulation of peddepod function can be applied both in cell culture and in therapeutic methods, in particular those that use the growth, control and maintenance of beta cells and possibly non-pancreatic cells, as well as in the control of development and maintenance of digestive tissues tract, spleen, lungs, genitourinary organs (for example, bladder) and other organs that develop from the primary intestine.

In an exemplary embodiment, the proposed method can be used in the treatment of hyperplastic and neoplastic diseases that affect pancreatic tissue, especially those where pathological proliferation of pancreatic cells is observed. For example, pancreatic cancer is characterized by pathological proliferation of pancreatic cells, which can eventually affect the insulin secretory 65 alteration of the pancreas. For example, in certain pancreatic hyperplasias, such as pancreatic carcinomas, hypoinsulinemia leads to beta-cell dysfunction or a decrease in islet cell mass. If aberrant ris, peddepod and ztooipepey signaling can be detected in the advanced stage of the disease, then the proposed inhibitors can be used to enhance tissue regeneration after antitumor therapy.

Moreover, the application of the signaling properties of peddepods in different variants may be useful as part of a strategy to transform/regenerate pancreatic tissue in both ip miigo and ip mimo. In one of the embodiments, in the proposed method, the discovery of the influence of ris, peddepod and ztooipepei on the process of regulating the development of pancreatic tissue is used. In general, this method can be used therapeutically to regulate the function of the pancreas after physical, chemical or pathological damage. In another 7th Embodiment, the proposed method can be used in cell culture, in particular, to enhance the primary production of implanted pancreatic cells. Manipulating the proliferation and differentiation of pancreatic tissue, for example by activating peadepods, may provide more precise control of the properties of the cultured tissue. In an example embodiment, the proposed method can be used both to increase the production of prosthetic formations requiring islet beta cells, and for use in encapsulation devices described in Aebrizspeg et al., US Pat.

Mo4892538, Aerizspeg, etc., US patent Mo5106627, IT, US patent Mo4391909 and bBepyop, US patent

Mo4353888|. Primary progenitor cells in pancreatic islets are multipotent and apparently co-activate all islet cell-specific genes from the time of their first appearance. During development, the expression of islet cell-specific hormones, such as insulin, is limited to the 2o expression pattern of mature islet cells. But the phenotype of mature islet cells is not stable in culture, as secondary appearance of embryonic features can be observed in mature beta cells. When using the proposed Ppeddepod antagonists, it is possible to regulate the direction of differentiation or the proliferative index of cells.

Moreover, manipulation of the regulation of the differentiated state of the pancreatic cell can be used in transplantation of an artificial pancreas to support implantation, vascularization, as well as ip mimo differentiation and support the transplanted cells. For example, i) manipulation of the function of pedeiod on tissue differentiation can be used as a means of maintaining the viability of the implant.

IVeiiizsi ei ai. (1997) YuOemeyurtepi 124;53)| reported that Zopis peddepod regulates the proliferation of lung mesenchymal cells. Thus, the proposed method can be used to regulate the regeneration process of lung tissue, for example, in the treatment of emphysema and respiratory distress syndrome.

I(Echiyka ei a/y. (1997) Viospet Viorpuz Kez Sottyp 238:658) reported that Zopis peddepod was expressed by M in human squamous lung carcinoma and adenocarcinoma cells. Zopis peddeipod expression was also detected in human squamous cell carcinoma lung tissue, but was absent in normal lung tissue in the same Me

From the patient himself. They also observed that Zopis pledeiod stimulated both the introduction of bromodeoxyuridine y- (BgaO) into carcinoma cells and their cell growth, while anti-5pPy-M inhibited cell growth. These results indicate that ris, peddepod and/or ztoipeped directly affect the process of cell growth in the lung tissue transformed in this way, and thus show that the proposed method can be used as part of the treatment process in lung carcinoma and adenocarcinoma, as well as in other proliferative "diseases in the lung epithelium. with c Many other tumors can be neutralized by treatment with the compounds proposed if there is evidence of . the influence of peddepods on the formation of such tumors, and the detected expression of peddeiod or its receptor in these cells? tissues during their development. The list of such tumors includes (but is not limited in any way) - tumors accompanied by Gorlin syndrome (for example, basal cell carcinoma, medulloblastoma, meningioma, others), tumors that are found in mice with knockout rsi genes (for example, hemangioma, rhabdomyosarcoma, -And others), tumors that are the result of amplification of the dii-1 gene (for example, glioblastoma, sarcoma, others), tumors that are associated with TCS8, a homologue of ris (for example, kidney carcinoma, thyroid carcinoma, others), tumors , se) which are related to Eh! (for example, bone cancer, others), tumors induced by ZPI (for example, lung cancer, -I chondrosarcoma, others) and other tumors (for example, breast cancer, urogenital cancer (for example, kidney, bladder, urethra, prostate, others), adrenal gland cancer, cancer of the digestive system (stomach, intestines, others). o In yet another embodiment of the proposed invention, compounds that contain peddepod antagonists can be 4) used to generate ip migo of skeletal tissues, for example, from skeletal stem cells, or for the treatment of diseases with a deficiency of skeletal tissues. The proposed invention is particularly directed to the use of Peddepyod antagonists to regulate the rate of chondrogenesis and/or osteogenesis. Under this expression "deficiency of skeletal tissues" is meant a disturbance in bones or other skeletal connective tissues in any cases when it is necessary to restore a bone or other connective tissue - and it does not matter, (F, what is the origin of this deficiency: the result of surgical intervention , removing a tumor, ulcer, taking material for implantation, other traumatic or degenerative conditions.

For example, the method according to the proposed invention can be used as an integral part of the therapeutic process to restore the function of cartilage in the connective tissue. Such methods are used, for example, in the elimination of defects or lesions of cartilage tissue due to degenerative changes, as it happens, for example, in arthritis, in mechanical disorders due to tissue trauma, such as displacement of torn meniscus tissue, meniscectomy, ligamentitis, malignant formations in the joint area , broken bones or hereditary diseases. The proposed method of repair can also be used for remodeling 65 of the cartilage matrix, for example, in plastic or reconstructive surgery, as well as in periodontal surgery. The proposed method can also be used in cases where it is necessary to improve a previously performed reparative operation, for example, after an operation on the meniscus, ligament or cartilage. Moreover, the method can be used to prevent the onset of symptoms or a more severe course of a degenerative disease if the treatment is applied immediately after the injury.

In one embodiment of the invention, the proposed method comprises treating damaged connective tissue with a therapeutically effective amount of a peddeipode antagonist, particularly an antagonist selective for Ipaiap peddepsode signal transduction, to regulate the cartilage repair response in the connective tissue by controlling the rate of differentiation and/or proliferation chondrocytes, permeable in the tissue. Such connective tissues as articular cartilage, interarticular cartilage, costal cartilage (connecting the true ribs to the sternum), 7/o Ligaments and tendons are particularly sensitive to treatment in reconstructive and/or regenerative therapies using the proposed method. As used herein, the term regenerative therapy includes both the treatment of degenerative conditions that have already progressed to the point where tissue damage is truly manifest, and the prophylactic treatment of tissue where the degenerative process is in its initial or even preclinical stages.

In an illustrative embodiment, the proposed method may be used as part of a therapeutic intervention to treat cartilage in a diarthrodial joint, such as a knee, ankle, finger or toe joint, or jaw joint. Treatment can be directed at the meniscus of the joint, the articular cartilage in the joint, or both. To further illustrate, the proposed method can be used to treat degenerative knee diseases that may occur as a result of traumatic injury (eg, sports injury or improperly sized clothing) or osteoarthritis. The proposed antagonists can be administered in the form of injections into the joint, for example, using an arthroscopic needle. In some examples, the agent to be administered may be in the form of a hydrogel or other slow-dissolving preparations described above to ensure prolonged and regular contact of the agent with the tissue being treated.

The invention further foresees the possibility of using the proposed method in the field of cartilage tissue transplantation and treatment using prosthetic means. But there are problems, for example, due to the fact that the properties of cartilage and fibrocartilage differ between different tissues: between articular, meniscal cartilage, i) ligaments and tendons, between two ends of the same ligament or tendon, and also between parts of the fabric on its surface and in depth. The zonal arrangement of these tissues may reflect a change in their mechanical properties, and it may happen that the implanted tissue, which has not differentiated under these conditions, does not show the ability to respond accordingly. For example, when meniscal cartilage is used to repair the anterior cruciate ligament, the tissue metaplasia into purely fibrous tissue. Due to the regulation of chondrogenesis, the proposed method may be particularly useful for this problem, helping to adaptively control the implanted cells in a new environment and efficiently accommodate hypertrophic chondrocytes from an earlier stage of tissue development. me)

Similarly, the proposed method can be applied both for increasing the generation of prosthetic cartilage formations and for their implantation. The need to improve treatment methods motivated scientific developments, the purpose of which was to create a new cartilage based on collagen-glucosaminoglycan structures (Ziope et al. (1990) Siip Opypor Keiai Kei 252: 129), isolated chondrocytes (Sgapae et al). (1989) In Opypor Caves 7:208; and Takidamla her a. (1987). :753; C) with mop Zspgoedeg ei a). (1991) ) Viotei Maieg Caves 25:329; Yes, yes, yes. (1993) ) Viotei Maieg Kev 27:11; and Masapii ei ai. US patent Mo5041,138). For example, chondrocytes can be grown in culture on capable and resorbable biocompatible highly porous bases made of polymers such as, for example, polyglycolic acid, polylactic acid, agar gel or other polymers that decompose over time due to the hydrolysis of the polymer base into indifferent monomers. Matrices are designed to provide adequate nutrition and gas exchange for cells until engraftment occurs. Cells can be cultured ip myo until adequate cell volume and density are achieved as a result of the development of cells intended for implantation. One of the advantages of matrices is that they can be given the shape that is needed in specific cases, so the final product is shaped very close to the shape of the patient's ear or nose (as an example), in addition, flexible matrices can be used in cases where where it is necessary to manipulate them during the operation itself, for example, on the joint. 4) In one embodiment of the proposed method, the implants are contacted with a peddepod antagonist during certain phases of the cultivation process in order to control the processes of chondrocyte differentiation and the formation of hypertrophic chondrocytes in the culture.

In another embodiment, the implanted device is treated with a peddepod antagonist to actively remodel the implant matrix and make it more suitable for its intended function. As indicated above in relation to (F) tissue grafts, artificial grafts suffer from the fact that it is impossible to obtain them from a place that would correspond to the actual mechanical conditions of the environment where the implanted matrix will function.

The ability to regulate chondrocytes in the matrix thanks to the proposed method allows the implant to acquire properties that are similar to the properties of the tissue it is supposed to replace.

In another embodiment, the proposed method can be used to strengthen the attachment of prosthetic devices. As an illustration, the method can be used in the implantation of periodontal prostheses, where the treatment of the surrounding connective tissue stimulates the formation of periodontal ligaments around the prosthesis.

In yet another embodiment, the proposed method can be used as part of a bone generation procedure 65 (osteogenesis) in an animal lacking such skeletal tissue. Ipaiiap peddepod is especially associated with hypertrophic chondrocytes, which are ultimately replaced by osteoblasts. For example, a destination

Neddeipod antagonist according to the proposed invention can be part of the method of regulating the process of bone loss in the subject. For example, drugs that contain a Ppeddepod-antagonist can be used to control endochondral ossification in a formation that is a "model" for ossification.

In yet another embodiment of the proposed invention, a peddepod antagonist can be used to regulate spermatogenesis. Peddepod proteins, especially OP, have been identified to be involved in the differentiation and/or proliferation and maintenance of testicular sperm cell development. Op expression is initiated in progenitor cells

Sertoli immediately after activation of 5Zhu (testicular determining gene) and is stored in the testes in adults.

Males are capable of sexual life, but are unable to have children if they lack mature sperm. 7/0 Studies of testicular development in different genetic environments demonstrate that OPP regulates both early and late stages of spermatogenesis. IViddood ei ai. (1996) Sigg Vio! 6:298). In a preferred embodiment, the pedopiod antagonist can be used as a contraceptive. Similarly, Ppeddepod antagonists of the present invention can potentially be used to modulate normal ovarian function.

The proposed method can also be widely used for the treatment or prevention of epithelial tissue disorders, as well as for cosmetic purposes. In general, the method may be characterized as comprising the step of administering to the animal an amount of pedopode antagonist effective to alter the growth state of the epithelial tissue being treated. Routes of administration, dosage, and regimen will vary and will depend on the epithelial tissue(s) being treated. For example, topical dosage forms will have advantages when the tissue being treated is epidermal tissue, such as skin or mucosal tissue.

A method that "accelerates wound healing" is one that causes wounds to heal faster than the same wounds without treatment. "Acceleration of wound healing" may also mean that the method regulates the proliferation and/or growth of, among other things, keratinocytes, or that the wound healing will be accompanied by the formation of fewer scars, the wounds will be less tight, with less collagen and more superficial. In some examples, "accelerating wound healing" may also mean that certain wound treatments will be more successful (eg, skin grafting) when used in conjunction with method i) of the present invention.

Despite significant progress in reconstructive surgical techniques, the formation of scars can be a significant obstacle to the return of normal function and appearance of the skin that has healed. This is especially so

This is true in cases where pathological scarring occurs, such as keloids or hypertrophic scars on the hands or face, which cause functional impairment and physical deformity. Under certain circumstances, such scars can cause psychosocial distress and lead to a life of economic deprivation. Wound healing includes stages of hemostasis, inflammation, proliferation and correction. In the proliferative phase, fibroblasts, endothelial and epithelial cells multiply. When using the proposed method, it is possible to control the process of proliferation of epithelial cells inside the wound and at its edges, in order to accelerate wound closure and/or reduce the formation of scars.

The treatment offered may be effective as part of a therapeutic plan for ulcers in the mouth or on surfaces close to the oral cavity, such as after radiation therapy or chemotherapy. Such ulcers appear, in most cases, within a few days after chemotherapy or radiation therapy. "

These ulcers usually begin as small, painful, irregularly shaped lesions, covered with a delicate gray plaque with a necrotic membrane and surrounded by inflammatory tissue. In many cases, lack of treatment leads to tissue proliferation on the periphery of lesions in connection with the inflammatory process. For example, the epithelium that surrounds an ulcer usually proliferates quite actively, as a result of which the continuity of the surface epithelium may be broken. These ulcers, due to their size and lack of epithelial integrity, can be

The entrance gate of the body for the development of a potential secondary infection in it. The usual consumption of food and water becomes an extremely painful process, and in the case of the spread of such ulcers along the digestive tract, diarrhea can occur with all the complications that are inherent to it. According to the proposed invention, the treatment of such ulcers, which includes the application of a peddeipod antagonist, can reduce the pathological proliferation and differentiation of the affected epithelium, and thus alleviate the severity of additional inflammatory processes.

The proposed method can also be used in the treatment of wounds due to dermatological diseases, such as lesions resulting from autoimmune disorders, for example, psoriasis. Atopic dermatitis 4) refers to skin injuries in the presence of allergies associated with an immune response to allergens such as pollen, food, insect venom and plant toxins.

In other embodiments, antiproliferative drugs of peddepod antagonists can be used for two TO to inhibit the proliferation of epithelial cells of the lens in order to prevent postoperative complications in extracapsular cataract extraction. Cataract is an eye disease that is difficult to control and

F) is treated, therefore many studies are devoted to the therapy of this disease. But now the treatment of cataract is surgical intervention. The surgical method has been used for a long time and many different methods of operations have been tried. Extracapsular cataract extraction has become the preferred method of cataract removal. Significant medical advantages of this technique in comparison with intracapsular extraction are the reduction of cases of cystoid macular edema and retinal detachment. Extracapsular extraction is also required for implantation of posterior chamber intraocular lenses, which is now considered the preferred method in most cases.

However, the disadvantage of extracapsular cataract extraction is a significant percentage of deterioration of the transparency of the posterior 65 capsule, which is often called secondary cataract, it can occur in almost 5095 patients during 3 years after the operation. The cause of secondary cataract is the proliferation of epithelial cells of the equatorial and anterior parts of the lens capsule, which remain after extracapsular cataract extraction. These cells proliferate and cause the appearance of Sommerling (Zottegiippo) rings, and together with fibroblasts, which also accumulate and deposit in the posterior capsule, cause opacification of the posterior capsule, which negatively affects visual function. Prevention of secondary cataract is better than its treatment. In order to inhibit the formation of secondary cataracts, the proposed method provides a means to inhibit the proliferation of epithelial cells of the remaining lenses. For example, such cells can be induced to remain quiescent by injecting, after removal of the lens, a solution containing a peddepod antagonist drug into the anterior chamber of the eye.

Moreover, the solution can be osmotically balanced to provide a minimum effective dose when 7/0 it is installed in the anterior chamber of the eye, thus inhibiting subcapsular epithelial growth with some specificity.

The proposed method can also be used in the treatment of diseases of the cornea of the eye, which are characterized by the proliferation of epithelial cells of the cornea, for example, in epithelial disorders of the cornea, such as epithelial proliferation or squamous cell carcinomas of the surface of the cornea. (emipe ei ai. (1997) 9 Meitsgovzsi 17:6277| showed that Peddepyod proteins can regulate mitogenesis and photoreceptor differentiation of the retina in vertebrates, and IPI is a possible factor from the pigment epithelium to increase the proliferation of retinal progenitor cells and photoreceptor differentiation. In addition,

MUepzep ei ai. (1997) JuOemeIortepi 124:363| showed that treatment of retinal cell cultures of perinatal mice with the amino-terminal fragment of Zopis peddenode protein results in an increase in the proportion of bromodeoxyuridine-containing cells in the total number of cells and in rod photoreceptors, amacrine cells, and Müller glial cells, so scientists believe that Bopis peddeiod promotes the proliferation of retinal progenitor cells. Thus, the proposed method can be used in the treatment of proliferative diseases of retinal cells and to regulate the differentiation of photoreceptors.

Another aspect of the proposed invention relates to the use of this method for controlling the growth of hair follicles. Hair basically contains keratin, a hard and insoluble protein; its strength is due to the disulfide bond with cystine. Each individual hair consists of a cylindrical rod and a root, which is i) placed in a follicle - an onion-shaped hole in the skin. The bottom of the follicle contains a finger-like growth called a papilla, which contains the connective tissue from which the hair grows, and through which blood vessels supply the cells with nourishment. The shaft is the part that protrudes through the surface of the skin, while the roots are the part of the hair that is embedded in the skin. The base of the root continues into the hair bulb, which is located on the papilla. The cells from which hair is formed grow in the bulb of the follicle; they are drawn out in the form of fibers when the follicle cells proliferate. The term "hair growth" refers to the formation and elongation of hair fibers by M division of cells.

As you know, the general hair cycle is divided into C stages: anagen, catagen and telogen. During the active (22) phase (anagen), papilla epidermal stem cells divide rapidly. Daughter cells move up and differentiate in the form of concentric layers of hair. The transition stage, catagen, is marked by the presence of a break in the mitosis of stem cells in the follicle. The resting stage is known as telogen, in which the hair remains on the scalp for several weeks before the new hair developing underneath pushes the resting shaft out of its follicle. From this model, it becomes clear that the greater the number of dividing stem cells that differentiate into hair cells, the more hair will grow from it. Accordingly, ways to speed up or slow down hair growth can be implemented thanks to . potentiation or inhibition, respectively, of the proliferation of these stem cells. a In some embodiments, the proposed method can be used to reduce hair growth in humans as opposed to removing it by trimming, shaving, or waxing. For example, the proposed method

It can be used in the treatment of trichosis, which is characterized by abnormally fast or dense growth of hair, for example, hypertrichosis. In an example embodiment, peddeiod antagonists may have been used for hirsutism, a disorder characterized by abnormal hairiness. This method can also provide the process of prolonging the depilation effect. Moreover, due to the fact that a peptide antagonist will often exhibit cytostatic rather than cytotoxic properties against epithelial cells, such drugs can be used to protect hair follicle cells from cytotoxic agents that require entry into the 5- phase of the cell cycle in order to cause, for example, hair loss after irradiation. Treatment by the proposed method can provide protection of hair follicle cells in such a way that the cells remain in a resting state, for example, by inhibiting the transition of cells into the C-phase and thus preventing mitotic catastrophe or programmed cell death. For example, peddepod antagonists can be used in patients receiving chemotherapy or radiation therapy, after which hair usually falls out. Inhibiting the progress of cellular

F) cycle during such therapy, the method of treatment offered may contribute to the protection of hair follicle cells from death, which may otherwise occur due to the activation of cell death programs.

After the end of the therapy, the treatment in this way can also be stopped with a corresponding decrease in the inhibition of the proliferation of follicular cells.

The proposed method can also be used in the treatment of folliculitis, such as decalcifying folliculitis, worm-like atrophoderma or keloid folliculitis. For example, peddeioad antagonist cosmetics can be applied to the affected areas to treat pseudofolliculitis, a chronic condition that occurs most often in the submandibular area and is associated with shaving, characterized by the presence of 65 erythematous papules and pustules with hidden hair.

In another aspect of the invention, the proposed method can be used to induce differentiation and/or inhibit the proliferation of tissue that originates from the epithelium. Such forms of these molecules can be the basis for differential therapy of hyperplastic or neoplastic conditions that arise in epithelial tissue.

For example, such drugs can be used to treat skin diseases where there is abnormal proliferation or growth of skin cells.

For example, pharmaceutical preparations according to the invention are directed to the treatment of hyperplastic epidermal conditions, such as keratosis, as well as to the treatment of neoplastic epidermal conditions characterized by a high rate of proliferation for various types of skin cancer, such as, for example, basal cell carcinoma or squamous cell carcinoma. The proposed method can also be used in the treatment of autoimmune diseases affecting the skin, especially in dermatological diseases in which there is pathological proliferation and/or keratinization of the epidermis, for example, caused by psoriasis or atopic dermatitis.

Many common skin diseases such as psoriasis, squamous cell carcinoma, keratoacanthoma, and actinic keratosis are characterized by localized abnormal proliferation and 7/5 growth. For example, in psoriasis, which is characterized by the presence of bran-like, red formations located above the surface of the skin, at the same time, as is known, keratinocytes proliferate much faster than normal, and the differentiation process does not take place completely.

In one of the embodiments, the drugs of the proposed invention meet the requirements in the treatment of dermatological chronic diseases that are associated with keratinization disorders that lead to 2o abnormal proliferation of skin cells, which diseases may have inflammatory or non-inflammatory components.

Illustratively, pedeiod antagonist therapeutic drugs, such as those that induce calmness or differentiation, can be used to treat various forms of psoriasis, whether cutaneous, mucosal, or nigella. Psoriasis, as described above, is typically characterized by epidermal keratinocytes that exhibit extreme proliferative activation and differentiation along the "regeneration" pathway. high school

Treatment with an antiproliferative embodiment of the proposed method can be used to curb pathological epidermal activation and create a basis for stable remission in the course of the disease. and)

A significant number of other keratotic lesions are also candidates for treatment using the proposed method. For example, actinic keratoses, which are superficial, inflammatory premalignant tumors that appear on areas of the skin exposed to the sun and exposed to radiation. Lesions are brown erythematous with different shades. Modern treatment includes excisional and cryosurgery. But these treatment methods are painful and often cause the formation of cosmetically unacceptable scars. at

Accordingly, the treatment of keratosis, such as actinic keratosis, may include applications, preferably M topical (local), of Peddepod antagonist compositions in an amount sufficient to inhibit hyperproliferation of the epidermal/epidermoid cells of the lesion. me)

Blackheads (acne) are another dermatological disease that can be treated in the way that is suggested. Acne vulgaris, for example, is a multifactorial disease that most often occurs in adolescents and young adults and is characterized by the appearance of lesions of inflammatory and non-inflammatory origin on the face and upper body. The main malformation that leads to acne vulgaris is hyperkeratosis of the duct of a hyperactive sebaceous gland. Hyperkeratosis blocks the normal motility of skin microorganisms and follicles, and thus stimulates the release of lipase by the bacteria Rhoripirasiegisht aspevz, bacteria from epidermal staphylococcus, as well as fungal bacteria Rigozrogyt omaIe. Treatment with use. an antiproliferative peddeiod antagonist, especially when used topically, can be useful in preventing transient phenomena in the duct, for example, hyperkeratosis, which leads to the formation of these lesions. Further treatment may include, for example, antibiotics, retinoids, and antiandrogens. -I The proposed invention also provides a method of treating various forms of dermatitis. Dermatitis is a descriptive term for poorly defined lesions that may be pruritic, erythematous, and scaly, with blisters, pus, pus, and crusts. These lesions appear for any of a wide range of reasons. The most typical dermatitis are atopic, contact and diaper dermatitis. For example, seborrheic dermatitis is chronic, usually with itching, dermatitis with erythematous, dry, moist or oily scales, and yellow crusts on various parts of the body, especially on the skull, with exfoliation of a significant 4) amount of dry scales. The proposed method can also be used in the treatment of congestive dermatitis, often chronic, usually eczematous dermatitis. Actinic dermatitis is dermatitis that develops as a result of radiation exposure, such as solar radiation, ultraviolet waves, or X-ray or gamma radiation. The method proposed according to the invention can be used in the treatment and/or prevention of certain symptoms of dermatitis, which is a consequence of unwanted proliferation of epithelial

F) cells. Such treatments for these different forms of dermatitis may also include topical (local) and systemic therapy: corticosteroids, anti-pruritic medications, and antibiotics.

For example, it is proposed to use this method to inhibit angiogenesis. Neddepod is known for stimulating angiogenesis. Matrigel scaffolds impregnated with peddenode protein and injected into mice show significant neovascularization, while Matrigel scaffolds not bearing peddeipods show comparatively little vascularization. Neddeiodine protein is also able to increase the vascularization of the normally avascular mouse cornea. The Ris-1 gene is expressed in normal vascular tissues, including aortic endothelial cells, vascular smooth muscle cells, aortic adventitia fibroblasts, and atrial and ventricular cardiomyocytes. These b5 tissues are also sensitive to peddepod protein. Treatment with exogenous peddepod leads to upregulation of ris-1 expression. In addition, peddeiod proteins stimulate the proliferation of vascular smooth muscle cells.

Neddenode proteins also cause fibroblasts to increase the expression of angiogenic growth factors such as MESE, BESE, Apd-1, and Apo-2. Finally, peddepod proteins are known to stimulate recovery from ischemic injury and collateral vessel formation.

Given that peddeiod stimulates angiogenesis, peddepod antagonists would be expected to act as inhibitors of angiogenesis, especially in situations where some level of peddepod signaling is required for angiogenesis.

Angiogenesis is the basis of many disorders. Long-term, irregular angiogenesis is observed in many diseases, tumor metastases and abnormal growth of endothelial cells. Blood vessels arising as a result of angiogenic processes support the pathological damage that occurs in these conditions. Various pathological conditions arising from uncontrolled angiogenesis are grouped together as angiogenesis-dependent or angiogenesis-associated diseases. Methods of treatment aimed at controlling angiogenesis processes can stop the development of such diseases or mitigate their course.

Diseases caused or supported by or associated with angiogenesis include /5 neovascular eye disease, age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal implant detachment, neovascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency, strain from the use of contact lenses, atopic keratitis, keratitis of the upper limb, pterygium, dry keratitis, xerodermatosis (Ziodheps, Sjogren's syndrome), pink acne (rosacea), phlyktenulosis keratitis, syphilis, mycobacteriosis, lipid degeneration, chemical burns, bacterial 2o ulcers, fungal ulcers, herpes simplex, shingles, protozoal infections, Kaposi's sarcoma, ulcer

Moraine, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarthritis, trauma, sarcoidosis, Wegener's granulomatosis, scleritis, Stevens-Johnson syndrome, conditions after radial keratotomy, chronic inflammatory osteoarthritis, ulcerative colitis and Crohn's disease, hemangiomas , Osler-Weber-Randeau disease and hereditary hemorrhagic telangiectasia. high school

In addition, angiogenesis plays an important role in the development of cancer. A tumor cannot grow without its blood supply with nutrients and cleaning of cellular waste. Tumors for which angiogenesis is important (8) include solid tumors such as rhabdomyosarcoma, retinoblastoma, Ewing's sarcoma, neuroblastoma, and osteosarcoma, and benign tumors such as acoustic neuroma, neurofibroma, trachoma, and pyogenic granuloma.

Angiogenic factors have been found to be associated with several solid tumors. Prevention of angiogenesis c c It could stop the growth of these tumors and prevent the disorders that would arise in the animal as a result of their existence. Angiogenesis is also associated with blood tumors, such as leukemia, any of various acute or chronic neoplastic diseases of the bone marrow, in which there is an uncontrolled proliferation of white blood cells, which is usually accompanied by anemia, deterioration of the blood coagulation process and an increase in lymph nodes, liver and spleen. Angiogenesis is believed to play a role in bone marrow abnormalities that lead to the development of tumors such as leukemia. Cha

In addition to tumor growth, angiogenesis is important in the development of metastases. First of all, angiogenesis is important in tumor vascularization, which allows cancer cells to enter the bloodstream and circulate in the body. Once tumor cells have left their primary site and found themselves in secondary, metastatic sites, the process of angiogenesis must begin before a new tumor can grow and spread. Such "

By preventing angiogenesis, it can lead to the prevention of tumor metastases and, possibly, the cessation of neoplastic growth at the site of the primary location of the tumor. . Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. and?" Angiogenesis is an important step in ovulation and also in blastula implantation after fertilization. Prevention of angiogenesis can be used to affect amenorrhea, block ovulation, or prevent blastula implantation. -I It is assumed that the invention may be useful in the treatment and/or prevention of respiratory distress syndrome (RDS) or other diseases resulting from inappropriate surface tension of the lungs. ik Respiratory distress syndrome occurs as a result of insufficient surfactant in the pulmonary alveoli. Lungs - And vertebrates contain surfactant, which is a complex mixture of lipids and proteins, it provides an increase in surface tension during inhalation and a decrease in tension during exhalation. During exhalation, surfactant reduces tension so that surface tension forces cannot cause alveoli to collapse. Aerated alveoli that did not collapse during exhalation ensure the exchange of oxygen and carbon dioxide between the blood and alveolar gases and require significantly less effort to fill with air during the next phase of inhalation. During inspiration, pulmonary surfactant increases surface tension as the alveolar surface area increases. The increase in surface tension in the expanded alveoli counteracts the overflow of its air space and sends the air that has already arrived to those alveoli that are not aerated so well, thus ensuring uniform aeration (F, lungs. ka Respiratory distress syndrome is especially common among premature babies Infants Pulmonary surfactant is normally synthesized very slowly until the last six weeks of fetal life.v Human infants born prematurely more than six weeks before the normal term are at high risk of being born with inadequate amounts of pulmonary surfactant and inadequate rates of synthesis.

The more premature the child was born, the more significant the surfactant deficiency can be. Significant surfactant deficiency can lead to respiratory failure that develops within minutes or hours after birth. Insufficiency of surfactant causes progressive collapse of alveoli 65 (atelectasis) due to a decrease in the ability of the lungs to expand, despite maximal efforts to inhale. As a result, an inadequate amount of oxygen enters the newborn's blood. RDS can also occur in adults, usually as a result of insufficient surfactant biosynthesis.

Lung tissues of premature babies show a high activity of the PPdeiod signaling pathway.

Inhibition of this pathway when using peddeipod antagonists enhances the formation of disc-shaped bodies and the expression of genes involved in surfactant biosynthesis. Disc-like bodies are subcellular structures associated with surfactant biosynthesis. Therefore, treatment of prematurely born children with the use of

Peddepiode antagonist should stimulate the biosynthesis of the sufractant and improve the condition of the child with ROD. In those cases when adult ENT is associated with activation of the peddepod signaling pathway, treatment with the use of

The antagonist's Neddenpod should also be effective. 70 It has also been suggested that the use of pedepiode antagonists may specifically target disorders in which the affected tissue and/or cells exhibit high activation of the pedepiode signaling pathway.

Gene expression is activated by a number of signaling pathways, including AII-1, DII-2, and DII-3. The expression of dii-1 most consistently correlates with the activity of peddeipod signaling in a wide range of tissues and disorders, while aiyi-3 is significantly less so. Effector genes have encoded transfer factors that activate the effect of many genes that are required to determine the full effect of peddiode signaling. Dii-3 genes encode transcription factors that activate the expression of many genes required to achieve the full effect of peddepod signaling. However, the transcription factor c1Ii-3 can also act as a repressor of Ppeddepsod gene-inducers, so the expression of diii-3 can lead to a decrease in the effect of the Peddepsod signaling pathway. Whether dii-3 will act as a transcriptional activator or as a repressor depends on post-translational events, and therefore it is expected that the methods of determining the activating form (as well as the repressing) O1Y-3 protein could be a reliable determination of the activation of the pediodo pathway. Dii-2 gene expression is expected to be a reliable marker of activation

Paddeipod of the way. The adii-1 gene is highly expressed in a large number of cancers, hyperplasias, and immature lungs and serves as a marker for the relative activation of the pedeiod pathway. Additionally, tissues such as immature lungs that have high expression of the gene are also strongly affected by Peddepod inhibitors. high school

Accordingly, it can be considered that the determination of gene expression can be used as an effective predictive tool for identification of tissues and disorders, which will be particularly useful in treatment with a peddeipod antagonist. and)

In preferred embodiments, action-1 expression levels are detected either by direct transcript detection or by detecting protein or activity levels. Transcripts can be determined using any of a wide range of techniques, which are mainly based on hybridization of probes for dii-1 transcripts or synthesized cDNA. Well-known techniques include Northern blotting, reverse-transcriptase PCR, and analysis of transcript levels using microcells (tisgoaggau apaiuses). Methods for determining the protein level include Western blotting, immunoprecipitation, two-dimensional electrophoresis in a polyacrylamide gel (20 5O5-RACE) M (it has advantages over the standard method, which determines the positions of its proteins), as well as mass spectroscopy. Mass spectroscopy can be combined with a series of purification steps to achieve high-quality identification of the levels of many different proteins in a given sample. Mass spectroscopy and two-dimensional electrophoresis 20 5ЮО5B-RACE can also be used to identify post-transcriptional modifications of proteins, which include proteolytic phenomena, the process of ubiquitylation, phosphorylation, lipid modification, and others. The activity of sia can also be determined by the method of analysis of binding to the DNA substrate or imago transcriptional activation of target promoters. The method of delaying the DNA sample in the gel, footprinting and "

The method of DNA protein cross-linking is a method that can be used to determine the presence of a protein capable of binding to these binding sites on DNA. . In better embodiments, the activity levels of the transcripts are measured, and when diseased or affected tissues show ? abnormally high activity levels, they are treated with a Ppeddepod antagonist. Immature lung tissue, lung cancers (eg, adenocarcinomas, bronchoalveolar adenocarcinomas, small cell carcinomas), breast cancers (eg, intraductal carcinomas, intralobular carcinomas, -I tubular carcinomas), prostate cancers (eg, adenocarcinomas), and benign prostatic hyperplasia - all they show a pronounced increase in the expression levels of dy in certain cases. Accordingly, expression levels of dii-1 are an effective diagnostic method of determining which of these tissues should be treated with a peddeipod-I antagonist. Additionally, there is some correlative evidence that urothelial cell cancers (eg, bladder cancer, other urogenital cancers) will also have elevated levels of dii-4 in certain cases. o For example, it is known that loss of heterozygosity on chromosome 9422 is characteristic of bladder cancer. Gene 4) ris-1 is located in this position, and ris-1 loss of function is probably a partial cause of hyperproliferation, as in many other types of cancer. Accordingly, such cancers could also exhibit high levels of ai expression and be amenable to treatment with a pedeiodine antagonist.

The expression of ris-1 and ris-2 is also activated by the peddeiod signaling pathway, but these genes are less significant than the action of genes as markers of activation of the peddeiod pathway. In some tissues, only ris-1 or only ris-2 is expressed, although

F) Peddepod pathway is very active. For example, in Ipaiap peddeipsoa testis development, the peddeipsoa pathway is active, but only ris-2 is expressed. Accordingly, these genes are individually independent as markers of activation of the peddeipod signaling pathway, although simultaneous determination of both genes is considered useful in indicating which tissues require peddeipod antagonist treatment.

Some diseases that can be treated by the proposed method are not specific to humans, such as scabies.

In yet another embodiment, the proposed method can be used in the treatment of human cancers, especially basal cell carcinomas and other types of tumors of epithelial tissues, such as skin. 65 For example, peddepod antagonists can be used in the proposed method as part of the treatment of basal cell nevus syndrome (BNS) and other human carcinomas, adenocarcinomas, sarcomas and the like.

In a preferred embodiment, the proposed method is used as part of a therapeutic or prophylactic regimen of therapy (or prevention) of basal cell carcinoma (BCC). Deregulation of signaling pathways

The pathway may be the main feature of basal cell carcinoma, which arises as a result of ris mutations.

Persistent overexpression of human ris mRNA has been described in tumors of familial and sporadic BCC, determined by ip hybridization. Mutations that inactivate ris would be expected to cause mutant Ris to be overexpressed because ris exhibits negative autoregulation. Previous research shows that overexpression of peddepod proteins can also lead to tumorigenesis. That zopis nonadepod (ZPP) plays a 7/0 role in mouse tumorigenesis was suggested by studies in which overexpression of ZPP in the skin of transgenic mice showed signs of SBCN, including various BCC-like epidermal proliferations on the skin surface after only a few days skin development. A mutation in the ZNAi gene of a person with BCC has also been described; it is believed that ZPP or other human NA genes may act as dominant oncogenes.

Sporadic ris mutations were also seen in BCC of otherwise normal individuals, among 5/5 of whom UV-induced mutations were present. In one recent study of sporadic BCC cases, among 15 tumors with ris mutations, 5 UV-induced mutations were found, with either C to T or C to TT substitutions.

Another recent analysis of sporadic ris mutations in BCC and neuroectodermal tumors showed a single C to T substitution in one of three cases of ris mutations found in BCC. See, for example, Sooagisii a). (1997) Zsiepse 277:1109-13; Hie ey aYi. (1997) Sapseg Caves 57:2369-72; Wow, wow. (1997) Zsiepse 276: 817-21; Hie ei a). (1997) Sepez Spgotovotez Sapseg 18: 305-9; Zyuope her a. (1996) Maishge 384:129-34; doppwop her ai. (1996)

Zsiepse 272:1668-711.

The proposed method can also be used in the treatment of patients with SBCN, for example, in order to prevent the development of BCC or other manifestations of the disease, which may be due to ris - loss of function,

Peddepyod - purchase of a function or ztooiyepaid - purchase of a function. Basal cell nevus syndrome is an extremely rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age. Patients with SBCN are highly susceptible to the development of these tumors; a large number of which appear in i) the second decade of life, mainly on areas of the skin exposed to the sun. This disease also causes numerous developmental abnormalities, including pathology of the ribs, head and face, sometimes polydactyly, syndactyly, non-union of the vertebral arches. They also develop numerous tumors of various types, in addition to BKK: ovarian and heart fibroids, cysts on the skin and jaws, as well as in the central nervous system - medulloblastomas and meningiomas. The proposed method can be used to prevent or treat these types of tumors in patients with and without SBCN. Studies of patients with SBCN show that they have both M genomic and sporadic mutations in the ris gene, suggesting that these mutations are the most likely cause of the disease. me)

In another aspect of the present invention, pharmaceutical preparations containing peddepod antagonists are provided. uh-

For use in the proposed method, peddepod antagonists can be easily selected for administration in a biologically acceptable medium, such as water, buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like) or also in the form of mixtures.

The optimum concentration of the active ingredient(s) in the selected medium can be determined empirically, according to techniques well known to medicinal chemists. As used herein, the term "biologically acceptable medium" includes any and all solvents, dispersible excipients, and the like that may meet the requirements of the desired route of administration of the intended pharmaceutical preparation. ;" The use of such media for pharmaceutically active substances is known to those skilled in the art. That is, the use of these media in pharmaceutical preparations according to the invention is possible, with the exception of those commonly known media or agents that are incompatible with the activity of the Peddepod antagonist. Such drug carriers that meet these requirements and compositions based on them with the inclusion of other proteins are given, for example, in (the book of Ketipdiopye RIagptaseciisa! ., OZA 1985)). These carriers include injectable "depot forms". -I Pharmaceutical preparations according to the invention may also include veterinary drugs and preparations, for example, 5p preparations of peddepoo antagonists for the treatment of livestock or domestic animals, for example, dogs. o Methods of administration may also be due to rechargeable and biodegradable devices. In 4) in recent years, many sustained-release polymers have been developed and tested, which provide controlled entry of drugs into the body, including protein biopharmaceuticals.

There are many biocompatible polymers (including hydrogels), including both biosoluble and other polymers, that can be used to form an implant with a sustained release of a peddepod antagonist at a specific target site. (F, Preparations according to the present invention can be administered orally, parenterally, topically or rectally. Of course, they are taken in a form that is suitable for each route of administration. For example, they are administered in the form of tablets or capsules, by injection, inhalation, eye lotions, ointments, suppositories, plasters with bo Controlled release of the drug, and the like administration by injection, infusion or inhalation; topically as a lotion or ointment; rectally - by means of suppositories. Oral and local (external) administration are considered preferable.

The terms "parenteral administration" and "parenteral administration" as used herein refer to routes of administration other than enteral or external administration, and such administration is usually by injection, including, but not limited to, intravenous, intramuscular, intra-arterial , intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal,

transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastem injections and infusions.

The terms "systemic administration," "administered systemically," "peripherally administered," and "peripherally administered" as used herein refer to administration of a compound, drug, or other substance other than directly into the central nervous system, such that said compound, therapeutic the drug or other substance is administered to other systems of the patient, and thus may be subject to metabolism and other similar processes, for example, when administered subcutaneously.

These compounds can be administered to humans and other animals for therapeutic treatment by any acceptable 7/0 route of administration, for example, orally, nasally, as a spray, rectally, intravaginally, parenterally, intracisternally, and topically, either as powders, ointments, or drops, and transbuccally and sublingually.

Regardless of the chosen route of administration, the components of the present invention, which can be used in a suitable hydrated form, and/or pharmaceutical compositions of the present invention, are prepared as part of /5 preparations in the form of pharmaceutically acceptable dosage forms, as described below or by other traditional methods known to specialists.

Practical dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied, in order to provide an amount of the active ingredient that is effective to achieve the desired therapeutic response for the particular patient, composition and route of administration, without possible toxic effects on the patient.

The dosage level selected will depend on many factors, including the activity of the particular compound of the present invention used, or its ester, salt or amide, route of administration, time of administration, rate of elimination of the particular compound used, duration of treatment, presence of other drugs, compounds and/or or substances that are used in combination with the particular peddeipod sc antagonist being used, the age, sex, weight, condition and general health and previous medical history of the patient being treated, and other similar factors well known to those skilled in the art in the medical field. and)

A physician or veterinarian of ordinary skill in the art can readily determine the need for use and prescribe an effective amount of the pharmaceutical composition. For example, a physician or veterinarian may begin by prescribing lower doses of the compounds of the invention in a pharmaceutical composition than is necessary to achieve a therapeutic effect, and then gradually increase the dosage until the desired effect is achieved. i am

Of course, an adequate daily dose of the compound according to the invention is the amount of the compound that is the lowest dose that provides a therapeutic effect. Such an effective dose will, as a rule, depend on the above factors. In general, dosages of the compounds of the invention for intravenous, intracerebroventricular, and subcutaneous administration to a patient will range from about 0.0001 to about 10 .mu.g per 1 kilogram of the patient's body weight per day.

If necessary, the effective daily dose of the active compound according to the invention can be administered in the form of two, three, four, five, six or more subdoses, which are administered separately at appropriate intervals throughout the day, possibly in standard dosage forms. "

It is believed that the term "treatment" includes prevention, therapy and the recovery process. c c The patient receiving such treatment may be any animal in need thereof, including primates such as humans and other mammals such as horses, cattle, pigs and sheep; home;" poultry and domestic animals.

The compound of the invention can be administered as such or in mixtures with pharmaceutically acceptable and/or sterile carriers, and they can also be administered in combination with other antimicrobial agents, such as -I penicillins, cephalosporins, aminoglycosides and glycopeptides. Such complex therapy includes sequential, simultaneous and separate administration of the active compound in such a way that the therapeutic effects from the first administration do not disappear completely at the next administration of the compound. -I M. Pharmaceutical compositions

Although the compound of the present invention can be administered by itself, it is preferable to administer such a compound in the form of a pharmaceutical preparation (composition). Peddepod antagonists according to the invention can be included in the composition of drugs intended for administration by any convenient route, for use in medicine or veterinary medicine. In some embodiments, the compound included in the pharmaceutical preparation may be active as such or may be a prodrug, for example, capable of being converted to an active compound under physiological conditions. 5B Thus, in accordance with yet another aspect of the present invention, there are provided pharmaceutically acceptable compositions containing a therapeutically effective amount of one or more of the compounds described above (F, formulated in the form of a preparation together with one or more than one pharmaceutically acceptable carrier (additive) and/or diluent As described in detail below, the pharmaceutical compositions of the present invention may be formulated as preparations specifically for administration in solid and liquid form, including preparations suitable for the following: 1) for oral administration, e.g. in the form of mixtures (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application on the tongue; 2) for parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection, for example, in the form of a sterile solution or suspension; 3) for external use, for example, in the form of a cream, ointment or spray applied to the skin; or 4) for intravaginal or rectal administration, 65 for example in the form of a pessary, cream or foam. However, in some embodiments, the compound of the invention can simply be dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is pyrogenic, that is, it does not raise the patient's body temperature.

The term "therapeutically effective amount" as used herein means that amount of a compound, substance, or composition containing a compound of the present invention that is effective to produce some desired therapeutic effect by overcoming the loss of ris function, the acquisition of peddiode function, or the acquisition of ztoieped function. at least in a subpopulation of cells in the animal and with this - to block the biological consequences of the indicated pathway in the treated cells, with a rational benefit/risk ratio used in any treatment in medicine.

The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms that, according to reasonable medical judgment, are suitable for use when contact with human and animal tissues occurs without exhibiting excessive toxicity, irritation, allergic reaction, or other problems or complications, with an appropriate rational benefit/risk ratio.

The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid excipient, diluent, excipient, solvent, or encapsulating material, used to carry or transport the proposed antagonists from one organ or body part to another organ or body part.

Each carrier must be "acceptable" in the sense that it must be compatible with the other ingredients of the drug and not cause harm to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include the following: 1) sugars such as lactose, glucose, and sucrose; 2) starches, such as corn and potato starch; 3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; 4) powdered tragacanth; 5) malt; b) gelatin; 7) talc; 8) such excipients as cocoa and wax for suppositories; 9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; 10) glycols, such as propylene glycol; 171) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; 12) esters, such as ethyl oleate and ethyl laurate; 13) agar; 14) buffer agents, such as magnesium hydroxide and i) aluminum hydroxide; 15) alginic acid; 16) pyrogenic water; 17) isotonic solution; 18) Ringer's solution; 19) ethyl alcohol; 20) phosphate buffer solutions; and 21) other non-toxic compatible substances used in pharmaceutical preparations. As noted above, in certain embodiments of the present invention, pedeiodo antagonists may contain basic functional groups, such as amino or alkylamino groups, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable M salts" in this context refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of this invention. These salts can be obtained during the final isolation and purification of the compounds according to the invention (22) or in a separate reaction of the purified compound in the form of its free base with a suitable organic or inorganic acid with the isolation of the salt formed in this way. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate. , lactobionate, lauryl sulfonate, etc. |see, for example, Vegade ei ai. (1997) "Rpagtaseciisa! zai(8", U. Rpapt. Zsi. 66: 1-19). "

Pharmaceutically acceptable salts of the proposed compounds include conventional non-toxic salts or quaternary ammonium salts of the compounds, for example, salts with non-toxic organic or inorganic acids. So, for example, such traditional non-toxic salts contain salts - derived from inorganic acids, such as hydrochloric, ;" hydrobromic, sulfuric, sulfamic, phosphoric, nitric acid, etc.; as well as salts obtained from organic acids, such as acetic, propionic, succinic, glycolic, stearic, malic, grape, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, -I sulfanilic, 2-acetoxybenzoic , fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic acid, etc. and, In other cases, the compounds of the present invention may contain one or more acidic functional -I groups and thus be capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in such cases refers to relatively non-toxic inorganic and organic addition salts of the base compounds of the present invention. These salts, 4) similarly, can be obtained separately in the process of final isolation and purification of the compounds or by separate reaction of the purified compound in the form of its free acid with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkaline or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, aluminum, etc. salts. Representative (F) organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, and ethanolamine, diethanolamine, piperazine, etc. (see, for example, Vegode ei ai., above).

The compositions may also contain humectants, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colors, release agents, coating agents and sweeteners, correctors and fragrances, preservatives and antioxidants.

Examples of pharmaceutically acceptable antioxidants include: 1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite, etc.; 2) antioxidants soluble in oils, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated 65 hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; C) metal-chelating substances, such as citric acid, ethylenediaminetetraacetic acid (EDTO), sorbitol, grape acid, phosphoric acid, etc.

The preparations according to the present invention contain preparations suitable for oral, nasal, external (including buccal and sublingual), rectal, intravaginal and/or parenteral administration. The preparations may be conveniently presented in the form of standard dosage forms and may be prepared by any method known in pharmacy. The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form will depend on the host organism being treated and the specific route of administration. The amount of active ingredient that can be combined with the carrier material for the preparation of a single dosage form, as a rule, will be such an amount of the compound that gives a therapeutic effect. Generally, in one hundred percent, this amount will be from about 170 percent to about 99 percent of the active ingredient, more preferably from about 5 to about 70 percent, most preferably from about 10 to about 30 percent.

Methods of preparing such preparations or compositions include the step of combining a compound of the present invention with a carrier and possibly with one or more auxiliary ingredients. As a rule, preparations are prepared by combining the compound according to the present invention with liquid carriers, or with finely divided solid carriers, or with both, to obtain a homogeneous product with a uniform distribution of components, and then, if necessary, add the product form .

Preparations according to the invention, suitable for oral administration, can be in the form of capsules, wafers, pills, tablets, lozenges (in which a base with a corrigent - usually sucrose or tragacanth is used), in the form of powders, granules, or in the form of a solution or suspension in aqueous or non-aqueous liquid, or in the form of a liquid emulsion of the oil-in-water or water-in-oil type, or in the form of an elixir or syrup, or in the form of lozenges (in which an inert base such as gelatin or glycerin, or sucrose or gum arabic), and/or in the form of mouthwashes and the like, each form containing a given amount of the compound of the invention as an active ingredient. The compound of the invention can also be administered as a bolus, medicated slurry or paste. with

In solid dosage forms of the invention intended for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more than one (8) pharmaceutically acceptable carrier, for example sodium citrate or calcium hydroorthophosphate, and/or with each of the following: 1) fillers or diluents, such as starches, lactose, sucrose, glucose, mannitol, or silicic acid; 2) binders, such as, for example, carboxymethylcellulose, alginates, with Gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; 3) humectants, such as glycerin; 4) leavening agents such as agar-agar, calcium carbonate, potato or cassava starch, alginic acid, some silicates and sodium carbonate; 5) agents slowing dissolution, for example, paraffin; 6) M absorption accelerators, such as, for example, quaternary ammonium compounds; 7) moisturizers, for example cetyl alcohol or glycerol monostearate; 8) absorbents, such as kaolin or bentonite clay; 9) lubricating substances, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and their mixtures; and 10) i- dyes. In the case of capsules, tablets and pills, pharmaceutical compositions may also contain buffering agents.

Soft and hard gelatin capsules can also be filled with solid compositions of this type, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols, etc. "

Tablets can be made by compression or molding, possibly with one or more than one auxiliary ingredient. Pressed tablets can be prepared using a binder (for example

And gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, etc.? disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfactant or dispersing agent. Molded tablets can be made by using a suitable apparatus to form a mixture of a powdery compound moistened with an inert liquid -I diluent.

Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills, and granules, may be scored or may be prepared with coatings and coatings, such as enteric coatings or other coatings well known in the art. pharmacy They can also be prepared in such a way as to provide a slow or controlled release of the active ingredient from them, o using, for example, hydroxypropyl methylcellulose in different ratios, which allows obtaining 4) the desired release profile, as well as other polymeric matrices, liposomes and/or microspheres. They can be sterilized, for example, by filtration through a filter that retains bacteria or by including sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or in any other sterile medium for injection immediately before use. These compositions may also contain silencing agents, and may have a composition in which only the active (F) ingredient is released, or, preferably, the release occurs in a specific area of the gastrointestinal tract, possibly with a delay. Examples of formulations for implementation that can be used are polymeric substances and waxes.

The active ingredient can also be presented in microencapsulated form, if appropriate, with one or more of the excipients described above.

Liquid dosage forms for oral administration of compounds according to the invention contain pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents traditionally used in the field, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl b5 alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cotton, peanut, corn, wheat germ, olive, castor, and sesame oils), glycerin, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters with sorbitan, and their mixtures .

In addition to inert diluents, oral compositions may also contain adjuvants such as

Moisturizers, emulsifiers and suspending agents, sweeteners, corrigents, dyes, fragrances and preservatives.

In addition to active compounds, suspensions may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan ethers, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as their mixtures. 70 It is known that sterols, such as cholesterol, form complexes with cyclodextrins. Thus, in the best embodiments, when the inhibitor is a steroid alkaloid, it can be included in the composition of the drug with cyclodextrins, such as α, β- and γ-cyclodextrin, dimethyl-p-cyclodextrin and 2-hydroxypropyl-D-cyclodextrin.

Formulations of the pharmaceutical compositions of the invention for rectal or intravaginal administration 75 may be presented in the form of a suppository which may be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers containing, for example, cocoa oil, polyethylene glycol, suppository wax, or salicylate, which is solid at room temperature but liquid at body temperature, and which will thus melt in the rectum or vagina and release the active Neddepsod antagonist.

Preparations of the present invention suitable for intravaginal administration also include pessaries, tampons, creams, gels, pastes, foams or aerosols containing carriers known in the art.

Dosage forms for external or transdermal administration of the compound according to the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, plasters and preparations for inhalation. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffer solutions or propellants that may be required.

Ointments, pastes, creams and gels, in addition to the active compound according to the invention, may also contain such excipients as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, siloxanes, bentonites, silicic acid, talc and zinc oxide or their mixtures.

Powders and sprays may contain, in addition to the compound according to the present invention, such excipients as lactose, talc, co-silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these substances. Sprays may additionally contain traditional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane. h-

Transdermal patches have the added advantage of providing controlled release of the compound of the invention into the body. Such dosage forms can be made by dissolving or dispersing peddeniod antagonists in a suitable medium. h-

Absorption enhancers can also be used to increase the flux of Nneddepod antagonists through the skin. The rate of such flow can be controlled either by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel. "

It is understood that eye preparations, eye ointments, powders, solutions, etc. are also included in the scope of this invention. - c Pharmaceutical compositions of the present invention suitable for oral administration contain one or more than one compound of the invention in combination with one or more of the following: pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders that can be diluted to obtain sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, dissolved substances that make the preparation isotonic with the blood of the intended recipient, suspending agents or thickeners.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention are water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, such as olive oil, and organic esters for injections, for example, ethyl oleate. The required fluidity can be maintained, for example, by using covering materials such as

Mn lecithin, in the case of dispersions - by maintaining the required particle size, as well as using se» surface-active substances.

These compositions may also contain adjuvants, such as preservatives, humectants, emulsifiers or dispersants. It is possible to prevent the action of microorganisms by including various antibacterial and antifungal agents, for example paraben, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include isotonicity agents in the composition, for example sugars, sodium chloride, etc. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate or gelatin.

In some cases, to prolong the effect of drugs, it is desirable to slow down the absorption of drugs from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance that has poor solubility in water. The rate of drug absorption will then depend on the rate of its dissolution, which, in turn, may depend on the size and shape of the crystals. Alternatively, the delayed absorption of the parenterally administered form of drugs is obtained by dissolving or suspending the drugs in an oil carrier.

Injectable depot forms are made by incorporating microencapsulated matrices with the proposed compounds 65 into biosoluble polymers such as polylactide-polyglycolide. The drug-to-polymer ratio, and given the nature of the particular polymer used, can control the rate of drug release. Examples of other biosoluble polymers include poly(orthoethers) and poly(anhydrides). Injectable depot forms are also prepared by entrapping drugs in liposomes or microemulsions compatible with body tissues.

When the compounds of the present invention are administered as pharmaceuticals to humans or animals, they may be administered as such or in the form of pharmaceutical compositions containing, for example, from 0.1 to 99,595 (preferably 0.5 to 9090) of the active ingredient in combination with pharmaceutically acceptable carrier.

Addition of the active compound according to the invention to animal food is preferably carried out by preparing a suitable preliminary mixture for feeding, containing an effective amount of the active compound, and including this preliminary mixture in the formed ration. 70 Alternatively, an intermediate concentrate or food additive containing the active ingredient can be mixed with food.

The method by which such feeding mixtures and formed rations can be prepared and administered is described in reference materials (for example, in "Arriedy Apita! Myshgyiop", MU. N. Egeedtap apa SO., Zap Rhapsizso, ).5.A., 1969 or "PmezvioskK Reedz apa Reeadipd" 0 apa V rookv, Sogmaiyiv, Oge., O. 5. A., 1977).

MI Schemes of synthesis and identification of active antagonists

The proposed steroid alkaloids and members of the same class can be readily obtained using the cross-coupling techniques of Suzuki, Style (She) and others.

Such coupling reactions are carried out under relatively mild conditions, and they assume the presence of a wide range of inactive functionalities. and. Combinatorial libraries

The compounds of the proposed invention, including libraries of variants having various representative classes of substituents, are within the purview of combinatorial chemistry and other similar synthetic schemes (see, for example, international publication PCT UUO 94/08051). As a result, large libraries of related compounds, such as the diverse compound libraries presented above, can be rapidly screened using high-throughput assays to identify potential lead compounds that represent peddepod antagonists, as well as to clarify specificity, toxicity and/or cytotoxic-kinetic characteristics of the leader compound. For example, biological activity assays of ris, peddepod, or ztoiPeped, which i) can also be done using ris loss-of-function, peddepod-gain, or ztoi-Peped cells, can be used to screen a library of proposed compounds to identify compounds that have the activity of an agonist in relation to ris or the activity of an antagonist in relation to peddeipods or ztoopepeds.

By way of illustration only, a combinatorial library for purposes of the present invention is a mixture of related compounds that can be co-screened for a desired property. Preparation of many similar compounds in one reaction mixture significantly reduces the number of screening processes that need to be performed and simplifies them. Screening for suitable physical properties can be done in traditional ways. me)

Diversity in such a library can be created at many different levels. For example, aryl groups of the substrate used in combinatorial reactions can be diverse in terms of the aryl group of the core, for example, have variations in the ring structure, tal" or can vary in relation to other substituents.

Many techniques for creating combinatorial libraries of small organic molecules, such as the proposed peddepod antagonists, are known in the art. |See, for example, Viopaeye ei a). (1995) Tgepaz ApaY. Spent 14:83 In the city; US patents 5359115 and 5362899 ApPutakh; US patent 5288514 Stage; publication of PCT UUO 94/08051 TSI ei a).; US patents 5736412 and 5712171 AgOtsShie; Spe ei a). (1994) UZHASZ 116: 2661; Keith ei aI. (1993) ZHAS5 115: 252; :from" publication of PCT UUO 92/10092, UO 93/09668 and MO 91/07087; and the publication of PCT UMO 93/20242 | egpeg her a). Accordingly, it is possible to synthesize a number of libraries containing approximately 100 to 1,000,000 or more diversomers of the proposed peddepod antagonists, which can be screened for a specific activity or property. -I In an exemplary embodiment, a library of diversomers representing candidates for peddeipod antagonists can be synthesized using a scheme adapted to the methods described in |Z ei ai., publication PCT UMO ik 94/08051), for example, using the linking them to the polymer granule through a group obtained by -I hydrolysis or photolysis, which may be present in one of the positions of candidate antagonists or the 5p substituent of the intermediate synthetic compound. According to the method of Style |ZNII ei a)/.), the library is synthesized on a set of granules, and each granule contains a set of labels identifying a specific diversomer on this 4) granule. Cells with loss of ris function, acquisition of peddepod function, or acquisition of peddepod function can be superimposed on this library on granules, for which a peddepod antagonist is selected. Diversomers can be released from the granules, for example, by hydrolysis.

The structures of the compounds used according to the invention make it easy to carry out their effective synthesis. The nature of these structures, as it is generally described by formulas I1-II, allows to carry out

F) combinatorial assembly of such compounds using some combination of groups Ag, 4, X, M and 7, as defined above. For example, these subunits can be attached to the ring using acylation or alkylation reactions. Most of such reactions, including those shown in Figures 11, 12, 151 16, are both very mild and very reliable, and thus quite suitable for combinatorial chemistry. Such combinatorial approaches can involve options for directions described below.

Many variations in the above and related pathways allow for the synthesis of a large variety of libraries of compounds that can be tested as inhibitors of peddepod function.

Preparation of characteristic compounds according to the proposed invention 65 a. Illustration of the synthesis scheme

Figures 1-31 show examples of synthetic schemes for the creation of pedeiodo antagonists suitable for the methods and compositions of the invention.

The conditions of the reactions illustrated in the schemes of Figures 1-31 are as follows: 1) KСНСМ, Mamn, toluene (Aggpeit-Rogesp, 1990, 40, 11, 1242 2) NeZO,, No, phlegm |Aggpeit-Rogesp, 1990 , 40, 11, 1242)

C) N»BO,, EN, phlegm | (Aggpeit-Rogzsp, 1990, 40, 11, 1242) 4) Maon, EON, phlegm 5) (Voe»O, 2m Maon, tnE 6) NOM, K/X, TNE |Megsk, international publication U/O 96/06609) 70 7) Ra-s, n», meon 8) -VyOMO, Syvg, NVg, No |U. Oh Spent 1977, 42, 2426) 9) AgV (OH)", RYAI(RR z), dioxane |U. Honey. Spent 1996, 39, 217-223). 1987, 109, 5478-5486) 12) AgzpViz, RI(RRPz)4, dioxane |U. At. Spent Zos. 1987, 109, 5478-5486) 13) KMpO,, Ru, H2O |U. Honey. Spent 1996, 39, 217-223) 14) maok., TNE 15) MazK., TNE 16) NUK. Kz, TNE 17) NOMO, Mav, |Adu. Risogipe Spet. 1965, 4, 1-30) 18) RYAD(OAs)", Man, ORRE, RYSN", KON |U. Oho. Spent 1997, 62, 5413-5418) 19) i. ХХ, EBM, СНоСи», etc. KuzH 20) OSI", sa, OME 21) SNOM", EBO s 22) Ad2O, Ma»SOz, Ma»Z»2Oz, BUT ITeigapeadgop GI ey. 1979, 26671 23) A902SRY, EBM, MeOnN (Ogo. zZup., 1970, 50, 77; U. At. Spet. os. 1987, 109, 5432 i) 24) PON, TNE-MeOonNn 25) (EU)R (ОСНСОК, ВЙ, ТНЕ 26) MebosСсСн(ВПО-:Р(РИ) з, benzene с зо 27) KOH or KOIVy 28) Base, ХСНо)пPSОВ о 29) ORRA, EBM, toluene |Zupipeziz 1985, 220) M 30 ) NOMO, NO 31) 505, SySI, NSI, No |Zupipeziv 1969, 1-10, 6) Me 32) Lawson's reagent, toluene |Teigapedhop Azut. 1996, 7, 12, 3553) i- 33) KOM, solvent 34) 3095 HO", crystallized SNUSOZN |Ne/m. Sleep Asia 1968, 349, 323) 35) triphosgene, СНоСИ» |Tegapeagop GI ek., 1996, 37, 8589) 36) (EYU)2P(OSNI ISO20O1-Rg, TNE, her. May « 37) RESOLUTIONS, MasNV(O)sSNz, OM5O (Zupipeziv 1987, 4981 - p. 38) Hgo, SNSIz or another solvent |Zupipeziv 1987, 498) and 39 ) You, VizZpsi "» 40) SIBOOTM5, SS, |Spet. Veg. 1995, 128, 575-580) 41) MmMeoOon-NSY, phlegm 42) AN, EBO or HIVN., EYN or VNZ-TNE (Teigapeadgop I! ey., 1996, 37, 8589) -I 43) Mas, EBM, SN»SI» |Teiganedgop I eyyi., 1996, 37, 8589) so 44) Ma»5O»z, NO |Teigapedgop GI ey., 1996 . ) Meiyi, TNE p» 49) tSRVA, SNoSI» 50) NOMO, SygO, Sts(MO»z)", NgO |U. Ogu. Spets. 1977, 42, 2053) 51) KUM, solvent 52) NOMO, Mab5 (5) SOEi, NO (Ogo. Zupii. 1947, 27, 811 o 53) NOK" or NZK,, SNhSI" 54) -VNYOS(OSI, EBM, MNz, TNE ko 55) ROBAMN, SNoSI", Mavn (OAs )" 56) P2KAMN, Meon/SsnazSoO2n, Mavnzsm in 57) P26OH, EOS, NOVI, PIEA, SNІСІ" or ОМЕ 58) P2OHN, NVTY, NOVI, OIEA, SNСІ" or ОЕМ 59) KBOKAMN, EOS, NOVI, OI EA, SNSI" or OME 60) KoKAMN, NVTY, NOVI, OIEA, SNSI" or OME 61) ROSI", Ru, SNSI" 65 62) РКАМНМСО, solvent 63) Р2ОС(ОСИ, EBM, solvent

64) K2СО5Н, EOS or НВТИ, NOVI, SIEA, СНСИ" or ОМЕ 65) ОХ, EBM, solvent 66) (СНзЗ)»С-М(СМ), ОМЕ, ЕОН |U. Honey. Spent 1994, 37, 57-66). )-O or I-SuzOnH, NVTY, NOVI, SIEA, SNhSI" or OME 70) Vos(Tt)-O or I-SuzH, mavnazsmMm, meon/SsnasSo»n |Zupipeziv 1975, 135-146) 71) 5- TI-M-Vos cysteinal, SISNSN-SI or TNE, Mavn(ogds)" |. Oh Spent 1996, 61, 3849-3862) 70 72) TRA, SNCI», EBZIN or (3:1:1) thioanisole/"ethanedithiol/YUM5 73) TRA, SNCI» 74) ORRA, EBM, toluene, NOSNSNoZISN" | Teigapeadgop I ey. 1984, 25, 35151 75) TVAE, TNE 76) Base, TTN or Vy5H 77) Base, CoX or K/X 78) tzMnNo, Meon/SnNUSoOn, Mavnzsm 79) MoN,, KOH 80) Rad(ava)z , P(o-(01)5, EMN", MasivVy, dioxane, K.MN" | Tegganeagop Hek. 1996, 37, 7181-7184). 81) Cyanamide. 82) EGtos-SI, sodium bicarbonate. 83) VYSOSI , sodium carbonate. 84) AIMOSOSI, pyridine. 85) Benzyl bromide, base. 86) Oxalyl chloride, ОМ5О. сч 87) KSOMN". 88) Carbonyl-diimidazole, neutral solvents (for example, OSM, OME, TNE, toluene). ( 8) 89) Thiocarbonyl-diimidazole, neutral solvents (for example, OSM, OME, TNE, toluene). 90) Cyanogen bromide, neutral solvents (for example, OSM, OME, TNE, toluene). 91) KSOCI, triethylamine with zo 92) EMNMN", EOS. 93) KOBSSOSI, EBM, OSM. yuyu 94) MON, pyridine |U.Nei. Spet., 1980, 607) M 95) Base, neutral solvents (for example, OSM, toluene, TNE). 96) NoMOV, EOS. me ) 97) KSBMN". part 98) KSOSNVIK, neutral solvents (for example, OSM, OME, TNE, toluene), (Ogo. Rgos. Rger. Ipiy., 1992, 24, 127). 99) SNM", NS. |Zupipeziv, 1993, 197). 100) МН.МНК, neutral solvents (for example, OSM, OME, TNE, toluene). « 101) K505SI, LOBSTER. Teiganeadhop I ey., 1993, 34, 2749). in p. 102) EBM, AH. U. Oh Spet., 1990, 55, 60371.

Y 103) MOSI or SI. U. Oho. Spet., 1990, 55, 3916). and?" 104) NaMON, neutral solvents (for example, OSM, OME, TNE, toluene). 105) KSSK, neutral solvents (OSM, TNE, toluene). 106) KSNSNKEK, neutral solvents (OSM, TNE, toluene). -I 107) NMON, NS. 108) Thiocarbonyl-diimidazole, 5iO»o or VEZOEB. |). Honey. Spet., 1996, 39, 52281. se) 109) Thiocarbonyl-dimidazole, OVO or OVM. U. Honey. Spet., 1996, 39, 52281. -I 110) NMO", NS. 111) SISNSSOOE CHOGO. Keasiyopzv, 1959, 10, 143). o 112) Morpholine phenamine (Yenshg. U. Med. Spet., 1982, 17, 27). syu 113) EEOSOSNESM 114) VKESOSNA"СО2Еи 115) Ma»5Oz 5B 116) Н-МСНАКСО EE 117) ЕЮ.ССНЕМСО

F) 118) VSMNMN". ka 119) КСОСО5Н, |u. Honey. Spet., 1995, 38, 37411. 120) SNO, KOAs. 60 121) 2-fluoronitrobenzene 122) ZpSi, EUN, OME. 123) ESNO, Mavnzsm, NoOAs. 124) chNZ, meon. 125) 2,4, 6-MezRy5O2MN". 65 126) EBMN, SNoSI" 127) ME2OS (0) SI, EBM, SNoSI"

128) K2MN», EOS, NOVT, EBM, SNSI» 129) OVO, RAISNZ 130) VosMnen(SNozTOSNoMN», EOS, NOVT, EBM, SNІСТ» 131) Р2МНОСНСО»Me, NVTI, NOVT, EBM, SNСІ» 132) VoOsМНneN( СНозТОСНоОМ», ГИНМО5, ТНЕ 133) K2МННСНСО»Me, Mavn(оОдс)», СисНОСНоСИ or TNE 134) К-МНСНоСН(ОЕО», НВТИ, NOVТ, EBM, СНиСИ» 135) Mavn(OAs)z, СиСНСНоСИ or TNE, AsonN. 70 136) Piperidine, OME. 137) RI(RazR)., VizZpN. 138) KSO5N, EOS, NOVT, EBM, OSM. 139) KEMN", neutral solvents. 140) ESNO, Mavnzsm, NOAs. 141) KMSO, solvent. 142) KSO5N, EOS or NVTY, NOVI, OIEA, SNSI" or OME. 143) KSOSI, triethylamine 144) К505СИ, EBM, СНоСи". 145) ZpSi, EUN, OME. 146) KMN", EOS, NOVI, OIEA, SNoSI" or OME. 147) Dibromoethane, EBM, СНХСІ» 148) Oxalyl chloride, neutral solvents. 149) PON, TNE-MeOH. 150) Carbonyl-diimidazole, neutral solvents (for example, OSM, OME, TNE, toluene). ch 151) EMN", EVM, SNosSi". 152) Basis, VH. i) 153) OV, RISNZ 154) ORRA, EBM, toluene |Zupipeziz 1985, 2201 155) BOSI», sa! OME. со зо 156) АН, Lewis acid (AlSys, ZpSi/X, TiSiId), СНосСи». 157) NOMSNKSO HER, neutral solvents. o 158) VosnyaMsSNEeSO»-N, EOS or NVTY, NOVI, OIEA, SNІСІ» or OME. i- 159) ТРА, СНоСи». r. Scripting analysis me)

There are many methods suitable for determining the ability of a compound to activate the function of ris or to inhibit the function of ztoopepei or pedepsode, many of which can be used in high-throughput formats. For many drug screening programs that test libraries of compounds and natural extracts, high-throughput methods are desirable to maximize the number of compounds studied in a given time period.

Accordingly, libraries of synthesized and natural products can be tested on other compounds that are "peddepoo antagonists." z c In addition to methods that do not use cells, the test compounds can be studied. cell-based methods. In one embodiment, cells having a loss-of-function phenotype of ris, gain-of-function and? Neddepod or gain-of-function peptide can be brought into contact with the test agent of interest, and this method evaluates, for example, the inhibition of cell proliferation in the presence of the test agent. -I Many gene products are involved in raisinped-mediated signal transduction, including raisned, transcription factors of the sybrii5 and pyegyrrhiz (si) family, the serine/threonine kinase Thysei (10), and the products of the genes ik soviaI-2, vtooipepei and BORI (zyrrgezzogo oi Tysed). -I Induction of cells by pedeiodine proteins involves a cascade of activation and inhibition of "downstream" effectors,

The end result is, in some cases, a marked change in gene transcription or translation. Potential transcriptional targets of peddeipod-mediated signal transmission are the raisnaid gene (Niaaido apa Ipopat, 4) 1990 YuemeIurtepi 110, 291-301; Magido ey a). 1996), and in vertebrates - homologues of the sirishv8 ipieggiriy5 gene

Ogovorpia, genes SI |Niyi ey a). (1994) Journal of Biochemistry 162: 402-413). It was shown that the expression of the raisney gene is induced in the cells of the limb bud and neural plate that respond to ZNAIP (|Magido ei ai. (1996) dv RMAZ 93: 9346-51; Magido ei a. (1996) Oemeiyurtepi 122: 1225-1233) . The genes encode transcription factors having DNA-binding domains of the "zinc finger" (Ogepis et al. (1990) Sepez 5 YuOem 4: 1053-1067; Kiplieg et al.

F) (1990) Mine! Sei| Vio! 10:634-642). It has been reported that in limb buds, the transcription of the ZI gene is up-regulated in response to peddeipods, while the transcription of the SiiZ gene is down-regulated in response to peddeiod induction (Magido et al. (1996) Oemeiortepi 122: 1225-1233). By selecting regulatory and transcriptional sequences from such target genes, for example, from the raisped or sia genes, which are responsible for the positive or negative regulation of these genes in response to peddepod signal transmission, and operably linking such promoters to the reporter gene, it is possible to obtain a transcription-based method that is sensitive to the ability of a particular test compound to modify signaling pathways mediated by

Peddepod. Expression of a reporter gene thus provides an evaluable screening tool for the development of compounds that act as peddenpoa antagonists.

In the methods according to the present invention, the final stage of the above-described cascade of events, i.e. modulation of transcription, is determined based on the reporter gene. Accordingly, in the practical application of one of the embodiments of the method, the construction with the reporter gene is built into the reagent cell to generate a detectable signal that depends on the loss of the ris function, the acquisition of the peddepod function, the acquisition of the ztooipepey function, or the stimulation of the ZPP itself. Quantitative reporter gene transcription can be measured using any suitable method known to those skilled in the art. For example, the expression of mRNA from a reporter gene can be detected using RNase protection or RNA-based polymerase chain reaction (PCR); or the protein produced by the reporter gene can be identified by its characteristic staining or biological activity. The amount of expression of the reporter is then compared to the amount of expression in either the same 7/0 cell in the absence of the test compound, or it can be compared to the amount of transcription in an essentially identical cell that lacks the target receptor protein.

Any statistically or otherwise significant decrease in the amount of transcription indicates that the test compound somehow activated the normal ris signal (or suppressed the gain-of-function signal

Peddepod or ztooiPpeped), that is, that the compound being tested is a potential antagonist of Neddenpsod.

E. Business Methods

One aspect of the present invention relates to a complex comprising a Peddepod antagonist, for example as described herein, for the treatment or prevention of basal cell carcinoma in a patient, preferably a human, according to instructions (written and/or graphical) describing the use of the composition for treatment or prevention basal cell carcinoma, and, if desired, a warning about possible side effects and 2 about the incompatibility of drugs with each other or drugs with food products.

Another aspect of the present invention relates to a complex comprising a peddepod antagonist, e.g., also as described herein, in which the peddepod antagonist is used as part of a therapeutic or cosmetic application as described above (e.g., nerve tissue regulation, bone and cartilage formation and regeneration, regulation of spermatogenesis, regulation of smooth muscles, regulation of lungs, liver and other tissues originating from the primary intestine, regulation of hematopoietic function and regulation of skin and hair growth), by a patient, preferably a person, according to instructions (written and/or graphic ), describing i) therapeutic or cosmetic use and, if desired, warnings about possible side effects and incompatibility of drugs with each other or drugs with food products.

In addition, the invention provides a method of conducting a pharmaceutical business, which includes: (a) the production of a pharmaceutical preparation containing a sterile pharmaceutical filler and a peddeipod antagonist; and (B) marketing (e.g., providing promotional and/or informative presentations of the product (such as demonstrations, telemarketing and lectures), products (such as trial drug samples), and/or M documentation (including promotional leaflets, brochures, websites, posters, etc.)) for workers and health care institutions, such as doctors, hospitals, clinics, etc., of the benefits of using a pharmaceutical drug for the treatment and me) prevention of basal cell carcinoma. uh-

Another aspect of the proposed invention relates to a method of conducting a pharmaceutical business, comprising: (a) manufacturing a pharmaceutical preparation containing a sterile pharmaceutical excipient and

Neddeipod antagonist; and (5) marketing (e.g., providing promotional and/or informative presentations (such as demonstrations, telemarketing and lectures), products (such as drug trials), and Mabo documentation (including flyers, brochures, websites , posters, etc.)) for health care workers and institutions, such as doctors, hospitals, clinics, etc., of the benefits of using a pharmaceutical preparation as part of a therapeutic or cosmetic application, such as regulation;" nerve tissues, formation and regeneration of bone and cartilage, regulation of spermatogenesis, regulation of smooth muscles, regulation of lungs, liver and other organs originating from the primary intestine, regulation of hematopoietic function and regulation of skin and hair growth. -I Another aspect of the present invention relates to a method of conducting a pharmaceutical business, which includes: (a) the production of a pharmaceutical preparation containing a sterile pharmaceutical filler and an antagonist of Neddepoo; and (b) marketing (e.g., providing promotional and/or informative presentations (such as demonstrations, telemarketing and lectures), products (such as trial drug samples), and/or 5r documentation (including promotional leaflets , brochures, websites, posters, etc.)) for workers and health care institutions, such as doctors, hospitals, clinics, etc., about the benefits of using a pharmaceutical preparation as part of a therapeutic or cosmetic application, such as regulation of nervous tissues, formation and regeneration of bone and cartilage, regulation of spermatogenesis, regulation of smooth muscles, regulation of lungs, liver and other organs originating from the primary intestine, regulation of hematopoietic function and regulation of skin growth and

B HAIR.

Another aspect of the invention provides a method of conducting a pharmaceutical business, including: (a) providing

F) a distribution network for the sale of a pharmaceutical composition containing a sterile pharmaceutical filler and a peddepod antagonist; and (5) providing patients or physicians with instructional materials for the use of the pharmaceutical composition for the treatment and prevention of basal cell carcinoma. 60 Another aspect of the invention provides a method of conducting a pharmaceutical business, which includes: (a) providing a distribution network for the sale of a pharmaceutical composition containing a sterile pharmaceutical filler and a peddepod antagonist; and (5) providing patients or physicians with instructional materials for use of the pharmaceutical composition as part of a therapeutic or cosmetic application such as described above (eg, regulation of nervous tissue, bone and cartilage formation and regeneration, regulation of spermatogenesis, regulation of smooth muscle, regulation of lungs, liver and other organs originating from the primary gut, regulation of hematopoietic function and regulation of skin and hair growth).

Another aspect of the invention provides a method of conducting a pharmaceutical business, which includes: (a) determining the appropriate pharmaceutical preparation and dosage of a peddepod antagonist for the treatment and prevention of basal cell carcinoma; (B) establishment of the therapeutic spectrum of action of the pharmaceutical composition, taking into account the effectiveness and toxicity in animals; (c) provision of a distribution network for the sale of a pharmaceutical composition that has an acceptable therapeutic spectrum; and, optionally, (a) providing a sales force to sell the drug to health care facilities.

Another aspect of the invention provides a method of conducting a pharmaceutical business, which includes: (a) determining the appropriate pharmaceutical preparation and dosage of a pedeiodine antagonist for the treatment and prevention of the medical or cosmetic condition described above; (B) establishing the therapeutic spectrum of action of the pharmaceutical composition, taking into account the effectiveness and toxicity in animals in the treatment or prevention of the specified conditions; (c) provision of a distribution network for the sale of a pharmaceutical composition having an acceptable therapeutic spectrum for the treatment or prevention of said conditions; and, optionally, (a) providing a sales force to sell the drug to health care facilities for the treatment or prevention of /5 specified conditions.

Another aspect of the invention provides a method of conducting a pharmaceutical business, which includes: (a) determining the appropriate pharmaceutical composition and dosage of a pedeiodine antagonist for the treatment and prevention of basal cell carcinoma; and (B) granting a third party license rights to further develop and sell the pharmaceutical composition.

Another aspect of the invention provides a method of conducting a pharmaceutical business, which includes: (a) determining the appropriate pharmaceutical composition and dosage of a pedopiode antagonist for the treatment and prevention of the medical or cosmetic condition described above; and (b) providing a third party with licensing rights for the further development and sale of a pharmaceutical composition for the treatment and prevention of the condition described above.

The invention described herein in its general form will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the present invention, and are not intended to limit the invention. and)

Synthesis of exemplar inhibitors sosi Ivoyu o, CH.SI», pyriline C 2 ovono A

Cl. AND

Mn, at 1 -9 and - mo, . Ge powder "

SNUSOSN. toluene (1:23, 125 "С, 1 hour with s and 2" s

C From the end groups 83, DHM - 45 and: 5 so 2 n. t A, sia 0 . - ome Y. . entry 8) " I o t- in - CO me e y 3 4 E 5 (F, Direction 1 ka General methods. Unless otherwise indicated, all reactions were carried out in glassware, dried in the oven, using solvents for HPLC (high-efficiency (liquid chromatography). Purification of the compounds was carried out on silica gel Ziis de!

Niazp! Mazvieg of the II company reached Spgotaidgarpa. Thin-layer chromatography (TLC) was performed on plates with silica gel KieseiIdeI - 60 254 (Megsk). The TIN-NMR spectrum was recorded on a VgyKeg 300 MMK spectrometer in deuterated solvents as an internal standard. Low-resolution mass spectrum data were obtained on the platform

Misgotazgz I C7. A Hewlett-Packard (Nemie 65 Raskaga) 1100 chromatograph was used to determine the purity (LC spectra). 2-chloro-5-nitro-M-(2-nitrophenyl) benzamide 1. To a solution of 2-nitrophenylamine (15.00 g, 109 mmol) in СНоСИи»

(9 Oml) was added pyridine (43 ml, 2.86 volumes) at room temperature with subsequent dropwise addition of 2-chloro-4-nitro-benzoyl chloride (27.40 g, 125 mmol), dissolved in CHCl (100 ml), at 02С for 45 minutes.

The reaction mixture was stirred for 3 hours at room temperature. Immediately after the end of the reaction (control by LCMS - liquid chromatography-mass spectrometry), the crude mixture was evaporated to dryness.

The brown residue was suspended in ethyl acetate and filtered - yield of the indicated compound (33g, 9495), yellow precipitate: TN NMR (SOCI5) 5 10.91 (in, 1H), 8.85 (а49, 1), 8.53 (arr a, 1H), 8.28-8.21 (t, 2H), 7.70 (aa, 1h), 7.64 (a, 1H), 7.25 (da, 1H); M5 (E5, positive mode): 363 (M. 1441)y322|M i 11. 3-(1H-benzoimidazol-2-yl)-4-chlorophenylamine 2. Iron powder (15.42 g, 280 mmol) was added to the amide solution 70.1 («15.00g, 46.7mmol) in toluene (125ml) and acetic acid (bbml). The mixture was kept at a temperature of 12590 for about 1 hour (control of the completion of the reaction by LCMS), and the resulting crude mixture was filtered through a cotton swab (to remove iron particles). The filtrate was evaporated to dryness, dissolved in EtOAc, washed with a saturated solution of MnSO 3 , then with brine, dried over M950O, filtered and concentrated under reduced pressure. The resulting brown solid was re-dissolved in CHCl 2 and a few drops of hexane were added to form a precipitate, which was collected by filtration. Output 2 (5.66g, 4295), beige precipitate: L1H NMR (СО5О0) 5 7.64 (broad 5, 2H), 7.30-7.26 (t, ЗН), 7.13 (arr 4, 1H), 6.84 (aa, 1H); M5 (E5, positive mode): 244 IM. -11.

Urea synthesis 3. Pyridine (0.12 mL, 1.41 mmol) was added to a solution of amine 2 (300 mg, 1.233 mmol) in CHClCl (3.0 mL). A solution of 3,5-dimethoxyphenyl isocyanate (253 mg, 1.4 mmol) in CHClCl (4.0 mL) was added dropwise at 09 20 and stirred for 3 hours at room temperature (monitoring the completion of the reaction by HPLC). 1 M solution of citric acid was added to inhibit the reaction, the organic phase was separated and evaporated to dryness.

The crude mixture was dissolved in E(Ac), washed with saturated Mancet solution, then brine, dried over Ma950O, filtered and concentrated under reduced pressure. The obtained crude product was purified by flash chromatography (E(OAc:hexane, gradient elution), yield C (40mg, 890): NMR (CO05300)5 8.05. SM 25 (arr 4, 1H), 7.82 (y, 1H), 7.81 (4, 1) 7.80 (broad c, 1H ), 7.67 (a, 1H), 7.45 (broad t, 2H), 6.83 (v, He) 1H), 6.82 (z, 1H), 6.33 (broad t, 1H) .

Synthesis of amide 4. A solution of 3,4,5-trimethoxybenzoyl chloride (272 mg, 1.18 mmol) in CHClCl (4.0 mL) was added to a solution of amine 2 (250 mg, 1.03 mmol) in CHClCl (33.00 mL) at 02С in nitrogen atmosphere. The reaction mixture was stirred overnight i) 30 at room temperature (monitoring the completion of the reaction by LCMS). The crude mixture was diluted with EtOAc to approximately 495 MeON, washed with saturated Manso solution with brine, dried over M95054, filtered and concentrated under reduced pressure. The obtained crude product was purified by LC-liquid chromatography, yield 4 (25mg, 690), colorless solid, ННМР du) (СО5О0) 5 8.31 (arr 4, tn), 7.98 (aa, 1Н) 7.76 -7.74 (t, 2H), 7.67 (y, 1H), 7.46-7.44 (t, 2H), 7.34 (wide 325 8, 2H); M5 (E5, positive mode): 438 (M. 11; РХ (1.15, purity 29596). -

Synthesis of thiourea 5. Pyridine (0.2 ml, 2.3 mmol) was added to a solution of amine 2 (25 mg, 1.03 mmol) in CHClCl (33.0 ml) at 02C with the subsequent dropwise addition of a solution of benzodioxaniisothiocyanate (228 mg, 1.18 mmol) in

СНоСИ, (4.00 ml). The reaction mixture was stirred overnight (monitoring the completion of the reaction by LCMS). 1 M citric acid solution was added to inhibit the reaction, the organic phase was separated and evaporated to dryness. Z7Z

The crude solid was diluted with EOAc, washed sequentially with saturated Manso solution and brine, dried over M950, filtered and concentrated under reduced pressure. The crude product obtained was purified by flash chromatography (30-50956 EtOAc in hexane), yield 5 (95mg, 2190), pale yellow solid: TN NMR (CO53O0) 5 7.71 (arr 4, 1H), 7, 55 (yes, 1H), 7.50 (wide in, 2H) 7.40 (a tn) 7.19-7.13 (wide t, 2H), 6.79 (in, 1H), 6.67 ( c, 2H) 4.08 (z, 4H); M5 (E5, positive mode): 438 Ma i-11; РХ о, - (0.941, purity 29896).

Synthesis of sulfonamide 6. Pyridine (0.2 ml, 2.3 mmol) was added to a solution of amine 2 (250 mg, 1.03 mmol) in (Ce)СНоСИ» (3.0 ml) at 02С with subsequent dropwise addition of phenylsulfonyl chloride (0.15 ml, 1 ,18 mmol) in -1 СНоСИ, (4.0 ml). The reaction mixture was stirred for 1 hour (monitoring the completion of the reaction by LCMS). A 1 M solution of citric acid was added to the crude reaction mixture, the organic phase was separated and evaporated to dryness. The crude residue was diluted with EtOAc, washed sequentially with saturated Manso solution and brine, dried over Ma95O.5, filtered and concentrated under reduced pressure.

The resulting crude mixture, containing mainly the bis-alkylated product, was purified by flash chromatography (1:39:60, MeOonH:EH2OAc:hexane), yield 6 (20mg, 595), colorless solid: NMR (CO3O0) 5 8, 01-7, 99 (wide 4, 2H), 7.97 (a, 1H) 7.95 (0, tn), 7.84 (arr a, 1), 7.76 - 7.74 (t, 4H ), 7.68 (y, 1H), 7.66 (a, 1H), 7.55 (da, 1H); M5 (E5, positive mode) 384 (MN, РХ (1.190, purity 299965). (F, Direction 2 name) 60 b5

MN. oh Kv lnonE s5-O. d And trembling

Mk, t Y Dt tovnsvt

I 1) 1252. todnna

Y Shk kichgi grup 23, DHM S

R

1 veat that I 40 - 2-(2-methyl-5-nitrophenyl)-1H-benzimidazole 7. Benzene-1,2-diamine (2.60g, 25mmol) and 2-methyl-5-nitrobenzoic acid (5, 0g, 27.bmol) was suspended in polyphosphoric acid (30 ml, 1 volume) and kept overnight at a temperature of 1402C. Immediately after the end of the reaction (control by LCMS), the crude green mixture was dissolved in water, made alkaline by Masson (pH-14) and extracted with E(Ac (x2). The organic phase was washed with saline solution, dried over Ma95O"), filtered and concentrated under reduced pressure, yield 7 (4.70g, 7490), orange-brown solid: TN NMR (CoO055O) 5 13.20 (8, 1H), 8.80 (arr a, 1H), 8.84 (ad, 1nH ), 7.89-7.87 (t, 2H), 7.74 (a, 1), 7.40-7.45 (t 2H), 2.90 (c, ЗН); M5 (E5, positive mode): 254 |M- 1).

3-(1H-benzimidazole-2-yl)-4-methylphenylamine 8. Iron powder (3.0g, 50.9mmol) was added to a solution of nitrobenzimidazole 7 (4.30g, 16b.mmol) in toluene (30ml) and acetic acid (ZOml), and the reaction mixture was kept at 12592 for about 1 hour (monitoring the completion of the reaction by TLC). and)

The resulting crude mixture was filtered through a cotton swab (to remove iron particles). The crude solution was evaporated to dryness, diluted with EOAc, washed with saturated Manso solution (up to pH-7), and the liquid phase was re-extracted with EOAc. The combined organic extracts were washed with brine, dried over «9 Ma50, filtered and concentrated under reduced pressure, yield 8 (2.709, 7390), brown solid: 1H NMR (C50650) 5 12.55 (v, 1H), 7.73 (a, 1), 7.57 (y, 1H), 7.16 (broad t, 2H), 7.09 (a, 1H), 7.01 (arr and a, 1Hn), 6, 70 (ad, 1H) 5.15 (wide in, 2H), 2.39 (in, ЗН); M5 (E5, positive mode): 224 |M'- 11. i-

Synthesis of amide 9. Benzimidazolephenylamine 8 (25mg, 1.12mmol) and 3-B5Bdimethoxybenzoyl chloride (246bmg, F 1.2mmol) were suspended in СНоСи» (10.00ml) and stirred overnight at room temperature.

The crude mixture was diluted with EtOAc, then washed with citric acid. The liquid phase was basified with Masson and extracted into EtOAc. The combined organic extracts were washed with brine, dried over Masson, filtered, and concentrated under reduced pressure to yield 9 as a colorless solid : TN NMR (С2О65О) 5 12.65 (in, 1Н), 10.35 (5, 71Н), 8.19 (in, 1Н), 7.86 (а, 1), 7.74 (а, 1 ), 7.61 (a, 1), 7.32 (a, « 1H), 7.42-7.32 (t, 2H), 7.20 (c, 2H), 6.79 (c, 1H ), 3.89 (in, 6H), 2.61 (8, ЗН); M5 (E5, positive mode): 388 40. |М -11; РХ (1.19, purity 29796). t s Synthesis of amide 10. Benzimidazolephenylamine 8 (25mg, 1.12mmol) and benzodioxane-4-carbonyl chloride (22mg, h 1.2mmol) were suspended in СНоСи» (10.00ml) and stirred overnight at room temperature.» The crude mixture was diluted with EtOAc, then washed citric acid. The liquid phase was basified with Masson and extracted into EtOAc. The combined organic extracts were washed with brine, dried over MazoO, filtered and concentrated concentrated under reduced pressure, yield 10 as a colorless solid: NMR NMR - (С2О065О) 5 12.55 (5, 1М), 10.49 (5, 1Н), 9.07 (arr a, tn), 8, 70 (da, yin), 8.25 (aa, tn), 8.15 (arr a, tn), o 7.12 (aa, tn), 7.64 (a, 1), 7.52-7 , 48 (t, 1H), 7.38 (a, 1), 7.31 (a, 1), 7.17-6.95 (t, 2H), 3.28 (c, ЗН);

M5 (E5, positive mode): 329 |M'- -11; PH (0.858, purity 297905). - Direction Z 50. and-th that On a

Gr vo, etc. nn, iya 1

AND

FROM

F) o) not with ; compound 1 2-(2-chloro-5-nitrophenyl)-1H-benzimidazole-5-carbonitrile 12. 2-amino-4-dianophenylamine (0.59, 3.7 mmol) and 2-chloro-4-nitrobenzaldehyde (1 ,39g, 7.52mmol) were dissolved separately in a minimal amount of acetic acid and mixed together at room temperature. The orange substance formed was filtered, washed with acetic acid and heptane until the filtrate became colorless. The solid matter isolated at this stage,

was intermediate 11 (b0Omg, 45965): NMR (C 506550) 5 8.99 (arr a, 1H), 8.91 (v, 1H), 8.28 (aa, tn), 7.84 (9 , 1H), 7.57 (c, 1H), 7.35 (ad, 1H), 6.75 (a, 1H), 6.36 (c, 2H); M5 (E5, positive mode): 301 (M "11.

Cyclization of intermediate 11: The resulting brown solid was suspended in acetic acid and kept at 5022 for 6 hours. The crude mixture was diluted with E(OAc, neutralized with a solution of MnSO»z, washed with brine, dried over Ma 5 O), filtered and concentrated under reduced pressure - yield 12 (200 mg, 99905), brown solid: IN NMR (С206550) 5; 13.40 (wide zv, 1H), 8.66 (arr a, 1n), 8.30 (da, 1), 8.20 (v, 1), 7.90 (a, 1), 7.76 (а, 1Н), 7.50 (0, 1Н); M5 (E5, positive mode): 299 |M'- -11.

Biological analyses

Identification of the lead compound/High-throughput screening analysis

Test compounds were dissolved in DMSO (dimethyl sulfoxide) to a concentration of 10 mM and stored at -2020. To activate the Neddepod pathway in the analyzed cells, an octylated (lipid-modified) form of the M-terminal fragment of the Zopis Neddeiod protein (OST-ZNN) was used. This M-terminal fragment of ZNN was obtained using bacteria. |See, for example, Tauiog EK, ei a!., Viospetivighu 2001, 40, 4359-711.

Compounds can be tested in the "O1i-II is" assay described below, using the 1071/2(512) cell line, where the cells contain a reporter construct corresponding to Neddeipod, in which the luciferase gene is used as a reporter gene. Thus, the signaling activity of the Neddepod pathway can be measured through the O1Y-I is-response.

10171/2(5312) cells were placed in a 9b-well microtiter plate (MTP) in the amount of 20,000 cells/well in complete medium (DMEM with the addition of 1095 FBS-fetal bovine serum). The plates were then placed in an incubator for overnight incubation (P/N) at 37 C and 5956 C. After 24 hours, the medium was replaced with a medium for luciferase assay (OMEM with the addition of 0.596 FBS). The compound was diluted in assay medium at a ratio of 3:1000 (approximately 300-fold), yielding a starting concentration of 259 ranging from approximately 0.0003 µM to 3 µM. Gee)

Then 15O0μl of each sample was introduced into the first wells (in three parallels). After that, 3-fold dilutions of samples were made in MTP to seven wells, which in total gives three parallels of seven dilutions for each compound. Then the protein ligand OST-ZNN was diluted in the medium for luciferase analysis and added to each well with a final concentration of 0.µg/ml. Then the tablets were again placed in the incubator for further incubation i) overnight (P/N) at 372С and 595С. After 24 hours, the tablets were removed from the incubator and the medium was aspirated/removed. Cells were washed once with buffer for analysis (phosphate buffer solution (FBR) with 1 mM Ma" and 1 mM Ca?g). Then 5 µl of buffer for analysis was added to each well. The reagent for luciferase analysis was prepared according to the manufacturer's instructions (set I isi Me manufactured by Raskaga) and added it in bbOmkl Fd) to each well. The tablets were incubated at room temperature (RT) for about 30 minutes, after which the signal was read, also at RT, using the Torsoitspi (Raskaga) device. -

Similar assays were performed using human cell lines (specifically, human embryonic palatal mesenchyme cells modified with the construct (i.e. as described above) in a culture medium consisting of minimal support medium (MEM)/sodium pyruvate aq./1090 "

FBI, and in assay medium containing MEM/sodium pyruvate aq./0.590 FBI. OST-ZNN, Z7Z t0 was added, obtaining a final concentration of 1 μg/ml. Compounds identified in the assays described above are shown in Figure 32. The activities of specific compounds are presented in the Table below (activities are based on results obtained in human cell-based assays, except for the activities for compounds E-M): 1 hoyov | Voohoov b 1yuyu6 oo t

Fo shi i i EI o o 1 | R is 7 ayu all o uyu vo

And Vo hide | 01 osv 5 ko 1yaoyuv | U1hoov

Kk "0.01 in "0.05 ohm | R m a | o ko tv ko ev vv to hide | renewed and renewed 1 koov mov oyu | ov kyu | вв ев о в см 5 о 1 увув ож | Work at 7 p.m.

F

7 AY 16 You 0hoyov t si 1 hoyov | o" ov 1 khov | R" kov « yu m yam | ihoov from the village

I" o" 1 hov | RY kov ayu vy - shchi - ATO 1 hoyov | V" hoov s" ov | o" hoov y syu"

Examples of effects of compounds on basal cell carcinoma (BCC)

BCC culture: Trial samples of basal cell carcinoma cells, taken by the scraping method, containing a certain amount of skin stroma, were cultivated in an organotypic culture, that is, placed in an air/liquid environment. BCC samples were injected with a solvent (sterile 0.896 Mass solution) or compound A, collected

F) cultures on the surface of a plastic mesh and incubated for three days (with or without the compound) in a medium suitable for long-term cultivation of human skin. After 3 days of cultivation, samples were subjected to routine histological examination. The characteristics of the morphological features of BCC, for example, islands of undifferentiated basal cells, in some cases with a characteristic peripheral arrangement of nuclei and gaps in the stroma, were preserved when BCC cells were cultured in this system. Gene 91 I, a major indicator of peddeipod signaling, remained active at high levels in untreated cultures, as determined by /3-labeled

RNE-probes. 65 Quantitative DNA hybridization: 7-μm sections of paraformaldehyde-fixed, paraffin-embedded tissue containing large basal cell islands were cleared, rehydrated, digested with proteinase K,

subjected to acetylation and hybridization with ZZP-labeled RNA probes overnight. After a high-quality post-hybridization wash, sections were exposed in the dark to a high-sensitivity RIozrpogitadeg (radioactive profiler) screen at room temperature for several hours. After manifestation, I ЗЗР| - the signal was scanned using the Zsapeg Susiope (RasKaga) scanner. Separate basal cell islets were isolated, the signal was quantified and expressed in units of OI Ch(aidia!PonE opiv)umm2 using Oriitsapi software. Control values were averaged and summed to 10095; the values obtained for the samples treated with compound A were expressed as 90 of the control values. The results are shown in Figure 33. 70 Immunohistochemical study of caspase-3. Programmed cell death or apoptosis is an organized process for the elimination of unwanted cells, which causes caspase activation, DNA fragmentation, membrane blebbing, cell compression, and phagocytosis. Apoptosis was analyzed by immunohistochemistry for active caspase-3, a marker that detects the early manifestation of apoptosis. BCC cell cultures were subjected to standard histological analysis, sections were deparaffinized, treated with blocking solution (|and incubated with primary antibody, then washed and incubated with enzyme-labeled secondary antibody according to the manufacturer's instructions. The specific signal was visualized by enzymatic detection of the antigen-antibody complex. Slides with processed sections were covered with coverslips and exposed.

The results are shown in Figure 34, with control tissue on the left and compound A on the right.

The effect of the proposed compounds on the cells of other cancer tumors

Colon cancer cells: 20,000 colon cancer cells were mixed with 10,000 1071(512) cells described above and the mixture was added to each of the 96 wells of the plate. The following day, the proposed compound and a control substance were added to the culture. After 72 hours of incubation, cells were diluted to measure luciferase activity. Compound A showed the ability to inhibit the activity of luciferase, which indirectly reflects the activity of the pedeiod signaling pathway, compared to the control substance in this test. Figure 35 shows the relative inhibition of Peddepod-induced activation in 512 cells for antibodies 1E6 and 5E1 and compound A, and the relative inhibition of endogenous Peddepod activity in colon cancer cells for these compounds.

Prostate and Bladder Cancer Cells: The results of the same analysis using cell lines PO-3, a prostate cancer cell line, and CT-4, a bladder cancer cell line, are presented in Figure 36.

The effect of the proposed compounds on other tissues

Figure 37 shows the probes and primers used to evaluate the effect of the proposed inhibitors on the expression of 1 I-1 and ZARON, using reverse transcriptase polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (PCR in real time) Takman F (Tadtap). Cells of palatine mesenchyme of the human embryo (PMEL) were grown in the presence of Octyl-ZNN and the proposed compounds for 24 hours. Full-length RNA was collected and used in a standard RT-PCR analysis to assess the expression levels of OS I-1 and SARON transcripts. Figure 38 shows the results of testing compound A, 0.003-1.0mMμM; compounds O, 0.003-1.0 µM; -, negative control (only YuOM5O); zhk, "positive control (Octyl-ZNN (F 1mg/mol, without inhibitor).

Cells homozygous for the absence of the raisped gene (ris-pii) were grown in the presence of the proposed compounds - c for 72-96 hours. Full-length RNA was collected and used in standard RT-PCR analysis to assess the expression levels of 51 I-1 and SARON transcripts. Figure 39 shows the results of the test: compound C, 1 and 1 omM; compound O, 1 and t0μM; OM5O, negative control.

Cells of palatine mesenchyme of the human embryo (PMEL) were grown in the presence of Octyl-ZNN and the proposed compounds for 24 hours. Full-length RNA was collected and converted into cDNA by standard reverse transcription (RT) reactions. cDNA was then used in real-time PCR analysis to assess the expression levels of 1I1-4 and SZARON transcripts. Figure 40 shows the test results: compound A, 0.001-0.3μM; compound O, 0.001-0.3μM; ОМ5О-), negative control; OST-ZNN(s), positive control. - and CSO-7 cells (lung fibroblast) were grown in the presence of octyl-ZNN and compound A for 24 hours. sl 50 Full-length RNA was collected and converted into cDNA by standard ZT reactions. The cDNA was then used to assess the expression levels of transcripts 1 I-1 and ZARON by real-time PCR. Figure 40 shows the following test results: compound A, 0.001-1.0μM; ОМ5О(-), negative control; OST-ZNN(s), positive control.

All references above are thereby references to this section.

Equivalents

Many equivalents to the specific embodiments of the invention described herein may be found or used by those skilled in the art through ordinary experimentation. Such equivalents are deemed to be covered by the following patent formula clauses. name) in

Claims (36)

The formula of the invention 1. The method of inhibiting the activation of the leodelod signal pathway in the cell, in which the cell is brought into contact with the leodelod antagonist in an amount sufficient to inhibit the transmission of the leodelod signal, which differs in that an organic molecule represented by the general formula (1) is used as a leadelod antagonist: b5 BA; The formula of Xe M is 5-4 x 70 where, as far as valency and stability allow, X and 7 are independently -Щ(К)-, -О-, -5-, - (КУ)М-М(К7)-, - OM(K7)- or simple connection; M is -F(50)-, -C(55)-, -"C(-MA)-, ZO" or 5O; A is O, 5 or MK,; C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring fused with the ring to which it is attached; Ag is a substituted or unsubstituted aryl or heteroaryl ring; Ku represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or cycloalkyl, including polycyclic groups; E»o represents from 0 to 4 substituents on the ring to which it is attached; KU, in each case independently, is H, lower alkyl, Y-cycloalkyl, --heterocyclyl, Y-aryl, Y-heteroaryl; Ka, in each case independently, is H, lower alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; and Y, in each case independently, is a chain containing from 0 to 8 units selected from CO, MK, OO and 5, where K, in each case independently, is H or lower alkyl. 2. The method according to claim 1, which differs in that at least one of 7 and X is not a simple bond. high school 3. The method according to claim 1, which differs in that X-U-7 contains amide, urea or sulfonamide. 4. The method according to claim 1, which differs in that X is selected from the group containing M(Ka)-, -O-, -5-, and preferably (o) is MH. 5. The method according to claim 1, which differs in that K. contains an aryl or heteroaryl ring, possibly substituted with 1-5 substituents. co 6. The method according to claim 5, which differs in that the substituents on K. are selected from the group consisting of nitro, halogen, cyano, lower alkyl, acylamino (in particular, Ka-C(-O)MH-), alkoxy, alkylamino , substituted or M unsubstituted cycloalkyl, heterocyclyl, aryl and heteroaryl fused to an oaryl or heteroaryl ring. 7. The method according to claim 1, which differs in that X and the ring containing A are placed on Ag in the meta-position (o) from each other. them 8. The method according to claim 1, which is characterized by the fact that Co is from 1 to 4 substituents selected from the group consisting of halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl group, thiocarbonyl, ketone, aldehyde . lower alkenyl, 9-Ka, U-ZN, U-MNo, protected forms of the above groups, or s-in any two Co", when they occur more than once in a cyclic or polycyclic structure, may be taken together with by the formation of 4-8-ene cycloalkyl, aryl or heteroaryl. :with" 9. The method according to claim 8, which is characterized by the fact that Co is from 1 to 4 substituents selected from the group consisting of halogen, cyano, nitro, alkoxy, amino, acylamino (for example, K 8-C(: O)MH-), substituted or unsubstituted cycloalkyl, heterocyclyl, aryl or heteroaryl co-condensed with (c, and substituted or -1 unsubstituted lower alkyl). 10. The method according to claim 1, which is characterized by the fact that the cell has a phenotype of loss of the ris function, acquisition of the leodeloo function or acquisition of the ztooiPpepayd function. - 11. The method according to claim 1, which is characterized by the fact that the leodeloo antagonist inhibits signal transduction mediated by the loss of function of ris, the acquisition of the function of rheodelod or the acquisition of the function of stooiiepei at EO 50), which is equal to 1 µM or less. "co 12. The method according to claim 1, which is characterized by the fact that the leodeloo antagonist inhibits signal transduction mediated by the loss of function of ris, the acquisition of the function of rheodelod or the acquisition of the function of ztooiyepei at an EO of 50) equal to 1 nM or less. 13. The method according to claim 1, which differs in that the cell is brought into contact with the antagonist leodeiod ip micgo. 14. The method according to claim 1, which differs in that the cell is brought into contact with the antagonist leodelod ip mimo. (F) 15. The method according to claim 1, which differs in that the antagonist leodelode is administered as part of a therapeutic or cosmetic application. 16. The method according to claim 15, which is characterized by the fact that the therapeutic or cosmetic application is selected from the group consisting of the regulation of nerve tissues, the formation and regeneration of bone and cartilage, the regulation of spermatogenesis, the regulation of smooth muscle, the regulation of lungs, liver and other organs originating from the primary intestine, regulation of hematopoietic function and regulation of skin and hair growth. 17. A method of inhibiting the activation of the leodelod signaling pathway in a cell, in which the cell is brought into contact with the leodelod antagonist in an amount sufficient to inhibit the transmission of the leoddegoo signal, which would differ in that the organic molecule represented by the general formula (II) is used as the leodelod antagonist : go A -- ha ; Formula II eg- x-y, 7 that pi where, as far as valency and stability allow, X and 7 are independently -Щ(К)-, -О-, -5-, - (КУ)М-М(К7)- , - OM(K7)- or simple connection; M is -F(0)-, -F(-5)-, -C(-MK)-, ZO" or 50; A is O, 5 or MK,; C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring fused to the ring to which it is attached; Ki is substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or cycloalkyl, including polycyclic groups; E»o represents from 0 to 4 substituents on the ring to which it is attached; Ez represents from 0 to 4 substituents on the ring to which it is attached; KU, in each case independently, is H, lower alkyl, Y-cycloalkyl, --heterocyclyl, Y-aryl, Y-heteroaryl; Ka, in each case independently, is H, lower alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; and Y, in each case independently, is a chain containing from 0 to 8 units selected from CO, MK, OO and 5, where K, in each case independently, is H or lower alkyl. Ha 18. The method according to claim 17, which is characterized by the fact that at least one of 7 and X is not a simple bond. 19. The method according to claim 17, which differs in that X-U-7 contains amide, urea or sulfonamide. at 20. The method according to claim 17, which is characterized by the fact that X is selected from the group containing -(Ka)-, -O-, -5-, and is preferably MH. 21. The method according to claim 17, which differs in that K. contains an aryl or heteroaryl ring possibly "9 substituted with 1-5 substituents. 22. The method according to claim 21, which is characterized by the fact that the substituents on K. are selected from the group consisting of nitro, halogen, cyano, lower alkyl, acylamino (for example, Ka-C(-O)MH-), alkoxy, alkylamino, substituted or unsubstituted cycloalkyl, heterocyclyl, aryl and heteroaryl fused to an oaryl or heteroaryl ring. b 23. The method according to claim 17, which is characterized in that Co represents from 1 to 4 substituents selected from the group consisting of halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl group, thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate, 4-Ka, « 4-OH, yu-lower alkyl, .- lower alkenyl, 9-Ka, U-ZN, U-MNo, protected forms of the above groups, or any two K», when they occur more than once in a cyclic or polycyclic structure, may be taken together with by the formation of 4-8-ene cycloalkyl, aryl or heteroaryl. c 24. The method according to claim 23, which is characterized by the fact that Co represents from 1 to 4 substituents selected from the group consisting of halogen, cyano, nitro, alkoxy, amino, acylamino, substituted or unsubstituted cycloalkyl, heterocyclyl , aryl or heteroaryl, co-condensed with C, and substituted or unsubstituted lower alkyl. -I 25. The method according to claim 17, which is characterized by the fact that the cell has a phenotype of the loss of the ris function, the acquisition of the leodeloo function, or the acquisition of the ztooiPpeped function. ish 26. The method according to claim 17, which is characterized by the fact that the antagonist leodelsoo inhibits signal transduction mediated by the loss of function of -I ris, the acquisition of the function of leodelod or the acquisition of the function of ztooipepei at an EHOO of 50, which is equal to 1 µM or less. i-th 27. The method according to claim 17, which is characterized by the fact that the antagonist leodelsoo inhibits signal transduction mediated by loss of function c» ris, acquisition of function reodelod or acquisition of function ztooiepei at EO 50) equal to 1 nM or less. 28. The method according to claim 17, which differs in that the cell is brought into contact with the antagonist leobdeilod ip micgo. 29. The method according to claim 17, which differs in that the cell is brought into contact with the antagonist leobdelod ip mimo. 30. The method according to claim 17, which is characterized by the fact that the antagonist leodelode is administered within the framework of therapeutic or cosmetic use. co 31. The method according to item ZO, which is characterized by the fact that the therapeutic or cosmetic application is selected from the group consisting of the regulation of nervous tissues, the formation and regeneration of bone and cartilage, the regulation of spermatogenesis, the regulation of smooth muscle, the regulation of the lungs, the liver and other organs originating from the primary intestine, regulation of hematopoietic function and regulation of skin and hair growth. 32. A pharmaceutical preparation containing a sterile pharmaceutical filler and a compound characterized by the fact that the compound is represented by the general formula (1): b5 BA; The formula of IR is the same as 70 where, as far as valency and stability allow, X and 7 are independently -М(К7)-, -О-, -5-, -(КУ)М-М(К7)-, - ОМЖ(К7)- or simple connection; M is -F(50)-, -F(0-5)-, -C(-MA)-, ZO" or 5O; A is O, 5 or MK,; C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring fused with the ring to which it is attached; Ag is a substituted or unsubstituted aryl or heteroaryl ring; Ku represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or cycloalkyl, including polycyclic groups; EC" represents from 0 to 4 substituents on the ring to which it is attached; KU, in each case independently, is H, lower alkyl, Y-cycloalkyl, --heterocyclyl, Y-aryl, Y-heteroaryl; Ka, in each case independently, is H, lower alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; and Y, in each case independently, is a chain containing from 0 to 8 units selected from CO, MK, OO and 5, where K, in each case independently, is H or lower alkyl. 33. A method of treatment or prevention of basal cell carcinoma, which differs in that the pharmaceutical preparation according to claim 32 is administered to the patient in an amount sufficient to inhibit the development of basal cell carcinoma. (at) 34. A pharmaceutical preparation containing a sterile pharmaceutical filler and a compound characterized by the fact that the compound is represented by the general formula (II): E. A -- v. ; Formula II so M - x-y, 7 -y Ф xy- K where, as valence and stability allow, X and 7 are independently -Щ(К)-, -О-, -5-, - (КУ)М -М(К7)-, - ОМ(К7)- or a simple bond; "M is -F(50)-, -C(55)-, -"C(-MA)-, ZO" or 5O; in c A there is O, 5 or MK,; C is a cycloalkyl, heterocyclic, aryl or heteroaryl ring fused with the ring to which it is attached; K; is H or substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl or cycloalkyl, including polycyclic groups; -I EC" represents from 0 to 4 substituents on the ring to which it is attached; Ez represents from 0 to 4 substituents on the ring to which it is attached; se) KU, in each case independently, is H, lower alkyl, Y-cycloalkyl, --heterocyclyl, Y-aryl, y-heteroaryl; -And Ka, in each case independently, is H, lower alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl; and Y, in each case independently, is a chain containing from 0 to 8 units selected from CO, MK, O 1 and 5, where K, in each case independently, is H or lower alkyl. 4) 35. A method of treatment or prevention of basal cell carcinoma, which is characterized by the fact that the pharmaceutical preparation according to claim 34 is administered to the patient in an amount that is sufficient to inhibit the development of basal cell carcinoma. 36. The method according to claims 1 or 17, which is characterized by the fact that the cell has a phenotype of loss of the function of ris, acquisition of the function of leodeloo or acquisition of the function of ztooiPpeped. name) 60 b5