Patent Application
20090099094
Somatostatin, a tetradecapeptide discovered by Bras(eau et al. (Science, 1973, 179:77-79), has been shown to have potent inhibitory effects on various secretory processes and cell proliferation in normal and neoplastic human tissues such as pituitary, pancreas and the gastrointestinal tract. Somatostatin also acts as a neuromodulator in the central nervous system. These biological effects of somatostatin, all inhibitory in nature, are elicited through a series of G protein coupled receptors, of which five different subtypes have been characteris(ed, hereinafter referred to as “SSTR-1”, “SSTR-2”, “SSTR-3”, “SSTR-4” and “SSTR-5” for each of the five receptors or generally and/or collectively as “SSTR” (Patel, Y. C., Front. Neuroendocrinol., 1999, 20:157-98; and Zatelli, M. C. et al., J. Endocrinol. Invest., 2004, 27 Suppl(6):168-70). These five subtypes have similar affinities for the endogenous somatostatin ligands but have differing distribution in various tissues. Somatostatin binds to the five distinct receptor subtypes with relatively high and equal affinity for each.
There is evidence that somatostatin regulates cell proliferation by arresting cell growth via SSTR-1, -2, -4 and -5 subtypes (Buscail, L. et al., Proc. Natl. Acad. Sci. USA, 1995, 92:1580-4; Buscail, L. et al., Proc. Natl. Acad. Sci. USA, 1994, 91:2315-9; Florio, T. et al., Mol. Endocrinol., 1999, 13:2437; and Sharma, K. et al., Mol. Endocrinol., 1999, 13:82-90) or by inducing apoptosis via SSTR-3 subtype (Sharma, K. et al., Mol. Endocrinol., 1996, 10:168896). Somatostatin and various analogues have been shown to inhibit normal and neoplastic cell proliferation in vitro and in vivo (Lamberts, S. W. et al., Endocrin. Rev., 1991, 12:450-82) via specific somatostatin receptors. (Patel, Y. C., Front Neuroendocrin., 1999, 20:157-98) and possibly different postreceptor actions (Weckbecker, G. et al., Pharmacol. Ther., 1993, 60:245-64; Bell, G. I. and Reisine, T., Trends Neurosci., 1993, 16:348; Patel, Y. C. et al., Biochem. Biophys. Res. Comm., 1994, 198:605-12; and Law, S. F. et al., Cell Signal, 1995, 7:1-8). In addition, there is evidence that distinct SSTR subtypes are expressed in normal and neoplastic human tissues (Virgolini, I. et al., Eur. J Clin. Invest., 1997, 27:645-7) conferring different tissue affinities for various somatostatin analogues and variable clinical response to their therapeutic effects.
Binding to the different types of SSTR subtypes has been associated with the treatment of various conditions and/or diseases. For example, the inhibition of growth hormone has been attributed to SSTR-2 (Raynor, et al., Molecular Pharmacol., 1993, 43:838; and Lloyd, et al., Am. J. Physiol., 1995, 268:G102) while the inhibition of insulin has been attributed to SSTR-5. Activation of SSTR-2 and SSTR-5 has been associated with growth hormone suppression and more particularly GH secreting adenomas (acromegaly) and TSH secreting adenomas. Activation of SSTR-2 but not SSTR-5 has been associated with treating prolactin secreting adenomas. Other indications associated with activation of the somatostatin receptor subtypes include inhibition of insulin and/or glucagon for treating diabetes mellitus, angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy, inhibition of gastric acid secretion and more particularly peptic ulcers, enterocutaneous and pancreaticocutaneous fistula, irritable bowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS related diarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitis and gastrointestinal hormone secreting tumors, treatment of cancer such as hepatoma, inhibition of angiogenesis, treatment of inflammatory disorders such as arthritis, retinopathy, chronic allograft rejection, angioplasty, preventing graft vessel and gastrointestinal bleeding. It is preferred to have an analog which is selective for the specific somatostatin receptor subtype or subtypes responsible for the desired biological response, thus reducing interaction with other receptor subtypes which could lead to undesirable side effects.
Somatostatin and its receptors are expressed in normal human parafollicular C cells. Medullary thyroid carcinoma (MTC) is a tumor originating from thyroid parafollicular C cells that produces somatostatin as well as calcitonin and several other peptides (Moreau, J.-P. et al., Metabolism, 1996, 45(8 Suppl.1):24-6). This hypersecretion can result in a syndrome very similar to carcinoid syndrome in which the patients may experience diarrhea, flushing, swelling, asthenia, weight loss, malaise and pain. MTC can occur sporadically or as part of the multiple endocrine neoplasia type-2 syndromes and familial MTC (Santoro, M. et al., J. Intern. Med., 1998, 243:505-8) representing 5-10% of all thyroid cancers (Cascon, A. et al., J. Clin. Endocrinol. Metab., 2005, 90:2127-30).
To date, the most effective treatment for MTC is total thyroidectomy associated with central neck lymph node dissection (Vitale, G. et al., Cancer, 2001, 91:1797-1808). While the complication rate for the initial surgery is reported to be relatively low at approximately 2%, second or additional neck dissections are associated with a very high rate of permanent damage to the patient in the form of hypoparathyroidism and laryngeal nerve palsy. In addition, persistent or recurrent disease is reported in 29-85% of the cases (Beressi, N. et al., Thyroid, 1998, 8:1039-44; Gimm, O. et al., World J. Surg., 1998, 22:562-8). The quality of life issues resulting from surgical complications are so severe that initial neck dissections are probably often performed too conservatively, accounting for the relatively high reported safety rate, and clinicians are often reluctant to perform additional surgeries. Radiotherapy and chemotherapy are of limited value in advanced MTC.
In 2006, the estimated prevalence in the United States and Europe for unresolved, symptomatic MTC was approximately 5,200 patients with an annual incidence rate in both territories of approximately 440 patients per year. There are currently no effective medical therapies for suppression of either calcitonin secretion or MTC proliferation following failed surgery, even though the symptoms, as well as the causative agents, are clear and readily measurable. Since there are no other approved treatments, any improvement would be beneficial, and as such, there is a major unmet medical need for a medical treatment for symptomatic residual medullary thyroid carcinoma following initial surgery. There is evidence of a highly variable SSTR expression pattern in MTC (Reubi, J. C. et al., Lab. Invest., 1991, 64:567-73; Zatelli, M. C. et al., Dig. Liver Dis., 2004, 36 Suppl. 1:S86-92). Previous in vitro studies of the effect of receptor subtype selective somatostatin analogs on the proliferation and calcitonin production/secretion by the MTC TT cell line, which expresses all SSTR subtypes, indicated that somatostatin and selective SSTR agonists do not all affect cell proliferation and calcitonin secretion the same (Zatelli, M. C. et al., J. Clin. Endocrinol. Metab., 2001, 86:2161-9; Zatelli, M. C. et al., Biochem. Biophys. Res. Commun., 2002, 297:828-34).
The aforementioned in vitro studies of the TT MTC cell line indicated that SSTR-2 ligands suppressed the proliferation of TT cells, while SSTR-5 ligands antagonis(ed and reversed the effects of activity at SSTR-2. It has been reported that activity at neither SSTR-2 nor SSTR-5, either individually or in combination, is effective in suppressing either calcitonin expression or secretion. These results may explain the lack of efficacy in suppressing calcitonin secretion of the two commercially available somatostatin analogs, lanreotide and octreotide, both of which have been shown to be highly potent SSTR-2 selective agonists with moderate SSTR-5 activity. In addition, treatment of MTC with either lanreotide or octreotide have had little effect on reducing tumour sis(e and/or increasing patient survival rate (Dies(, J. J. et al., J. Endocrinol. Invest., 2002, 25:7738; see also Vitale, G. et al., J. Clin. Endocrinol. Metab., 2000, 85:983-8).
Continuing studies have revealed that SSTR-1 analogs exhibit antiproliferative properties while also effectively suppressing calcitonin expression and secretion of MTC. It has also been observed that SSTR-1 selective analogs are also effective in suppressing the phosphorylation of cAMP response element binding protein, a transcription factor that facilitates a number of secretory pathways. These observations regarding the SSTR-1 receptor suggest that it too may effectively curtail the hypersecretory action of MTC cells. It has also been observed that SSTR-1 selective agonists are more effective in suppressing the hypersecretions MTC than native somatostatin which activates all of the receptor subtypes. As observed with the antagonistic interaction between the SSTR-2 and SSTR-5 receptors, certain receptor subtypes activated by native somatostatin may antagonis(e other subtypes.
It is apparent that distinct somatostatin receptor subtypes influence cell growth rate and hormone secretion differently, suggesting that the human body's response to a particular somatostatin analog is highly tissue specific. Development and assessment of SSTR subtype analogues selective on MTC cell growth would provide a useful tool for clinical application. The previously discussed in vitro studies conducted using MTC cell line TT provided valuable insight as to the ability of different somatostatin and somatostatin analogs to suppress the proliferation of and to reduce the secretion of calcitonin by MTC IT cells in vitro. To further extend the study and search for treatments for MTC, it would be beneficial to obtain and evaluate primary cultures of human thyroid medullary carcinoma cells, as well as other thyroid follicular cell tumors, removed at first surgery from actual patients.
As discussed herein, for the first time, the responsiveness of primary MTC tissue taken directly from human patients was studied in an effort to provide results useful to develop efficient and effective clinical treatments for human patients suffering from MTC. In particular, Applicants demonstrate: 1) that TT cell lines behave in a manner which is different than that of primary MTC cells isolated from tumor samples; and 2) that primary tumor samples from different patients also differ in their response to various somatostatin agonists. Taken together, this information provides a basis for determining optimum treatment plans for patients suffering from MTC, particularly post-operative reoccurrence of the disease, and its symptoms.
Applicants discovered that primary MTC tumor cells can be subdivided into two classifications according to inhibition of calcitonin secretion by the clinically available somatostatin analog “lanreotide: “lanreotide responsive”, in which calcitonin secretion was reduced by about 15% or more, or “lanreotide non-responsive”, in which calcitonin secretion was reduced by about 15% or less.
In the “lanreotide responsive” group, calcitonin secretion was significantly (P<0.05) reduced by lanreotide (−27%), the SSTR-1 selective agonist Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 (−17%), the SSTR-2 selective agonist c[Tic-Tyr-D-Trp-Lys-Abu-Phe] (−23%), and the SSTR-5 selective agonist D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 (−28%). A direct correlation was observed between the inhibitory effect of SSTR-2 interacting analogs and SSTR-2 mRNA expression levels. The SSTR-1 and SSTR-2 selective agonists had an additive inhibitory effect on calcitonin secretion (−38%) in this group. Little to no effect on cell viability was observed after treatment with any SSTR agonist. Treatment with a combination of an SSTR-1 agonist and an SSTR-2 agonist would be highly efficacious in suppressing calcitonin in the “lanreotide-responsive” subclass.
In the “lanreotide non-responsive” group, however, neither SSTR-2 nor SSTR-5 selective agonists alter calcitonin secretion. Only SSTR-1 selective agonists significantly reduce calcitonin secretion (−13%; P<0.05). In the “lanreotide non-responsive” group, cell viability was significantly (P<0.05) reduced by both lanreotide (−14%) and by the SSTR-2 selective agonist, c[Tic-Tyr-D-Trp-Lys-Abu-Phe] (−13%), but not by the SSTR-1 selective agonist, Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or by the SSTR-5 selective agonist, D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2.
CgA secretion was not affected by somatostatin analogs in any of the cultures.
In a first embodiment, the invention is directed to a method of determining the treatment for a patient suffering from medullary thyroid carcinoma comprising the steps of a) identifying the change in calcitonin secreted by a medullary thyroid carcinoma in response to a somatostatin agonist; and b) determining a treatment, wherein the determining of treatment is based upon the determined change in calcitonin secretion. In one aspect of the first embodiment, the method of identifying comprises: a) obtaining a sample of the medullary thyroid carcinoma from the patient; b) administering a somatostatin agonist to the carcinoma sample; and c) measuring the change in calcitonin secreted by the sample after administration.
The somatostatin agonist useful to practice the method of the first embodiment, and aspects thereof, includes SSTR-2 agonists or SSTR-2 selective agonists, or pharmaceutically acceptable salts thereof. Exemplary SSTR-2 agonists include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In one feature of the method of determining the treatment for a patient suffering from medullary thyroid carcinoma, the selection of treatment is based upon the change in calcitonin levels following treatment with an SSTR-2 agonist where a the choice of treatment for carcinomas or patients exhibiting a decrease in calcitonin secretion of less than about 15% (i.e., a “non-responder” carcinoma or patient) may differ than the choice of treatment for carcinomas or patients exhibiting a decrease in calcitonin secretion of greater than about 15% (i.e., a “responder” carcinoma or patient). In another aspect, the MTC patient in need of treatment and benefiting from the method of the instant invention has undergone at least one thyroidectomy.
In a second embodiment, the invention is directed to a method of decreasing the secretion of calcitonin from medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with an SSTR-1 agonist, an SSTR-2 agonist, an SSTR-5 agonist, or a combination of one or more SSTR-1 agonist and one or more SSTR-2 agonist, wherein the medullary carcinoma cells are determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof.
In a first aspect of the second embodiment, the SSTR-2 agonist used to determine the decrease in calcitonin levels is an SSTR-2 selective agonist or a pharmaceutically acceptable salt thereof. The SSTR-2 selective agonist, or pharmaceutically acceptable salt thereof, may have a Ki value of less than 5 nM or even less than 1 nM. Exemplary SSTR-2 agonists useful to determine the decrease in calcitonin levels include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr NH2, or pharmaceutically acceptable salts thereof. In one feature, the exemplary SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-N or a pharmaceutically acceptable salt thereof. In yet another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a second aspect of the second embodiment, the medullary thyroid carcinoma cells are contacted with an SSTR-1 selective agonist, an SSTR-2 selective, an SSTR-5 selective agonist, or a combination of one or more SSTR-1 selective agonist and one or more SSTR-2 selective agonist, or pharmaceutically acceptable salts thereof. In one feature, the SSTR-1, SSTR-2 and SSTR-5 selective agonists or pharmaceutically acceptable salt thereof, have a Ki value of less than 5 nM. In yet another feature, the SSTR-1, SSTR-2 and SSTR-5 selective agonists or pharmaceutically acceptable salt thereof, have a Ki value of less than 1 nM.
In a third aspect of the second embodiment, an SSTR-1 agonist or a pharmaceutically acceptable salt thereof is used to contact the medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. Exemplary SSTR-1 agonists include, but are not limited to, Caeg-c[D-Cys-3 Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-3 Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2, or Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In one feature, the exemplary SSTR-1 agonist is Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-1 agonist is Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In yet another feature, the exemplary SSTR-1 agonist is Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)Tyr-NH2 or a pharmaceutically acceptable salt thereof.
In a fourth aspect of the second embodiment, an SSTR-2 agonist or a pharmaceutically acceptable salt thereof is used to contact the contacted medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. Exemplary SSTR-2 agonists include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In yet another feature, the preferred exemplary SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. E another feature, the exemplary SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH or a pharmaceutically acceptable salt thereof.
In a fifth aspect of the second embodiment, an SSTR-5 agonist or a pharmaceutically acceptable salt thereof is used to contact the medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. Exemplary SSTR-5 agonists include, but are not limited to, D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or D-Phe-[Cys-Tyr(1)-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In one feature, the preferred exemplary SSTR-5 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-5 agonist is D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a sixth aspect of the second embodiment, a combination of an SSTR-1 and an SSTR-2 agonist, or pharmaceutically acceptable salts thereof, is used to contact the medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. Exemplary SSTR-1 agonists include, but are not limited to, Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bs(l-Tyr-NH2, Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2 or Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and exemplary SSTR-2 agonists include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a first feature of the sixth aspect of the second embodiment, the SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof, and the SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NM, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a preferred aspect of the immediately foregoing, the SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In another preferred aspect of the foregoing first feature, the SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH and the SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, or pharmaceutically acceptable salts thereof. In another preferred aspect of the foregoing first feature, the SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH and the SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In another aspect of the foregoing first feature, the SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe], or pharmaceutically acceptable salts thereof. In another aspect of the foregoing first feature, the SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In yet another aspect of the foregoing first feature, the SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a second feature of the sixth aspect of the second embodiment, the SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof and the SSTR-2 is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a preferred aspect of the immediately foregoing, the SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In yet another preferred aspect of the foregoing second feature, the SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, or pharmaceutically acceptable salts thereof. In yet another preferred aspect of the foregoing second feature, the SSTR-1 agonist is Aaeg-p-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In yet another aspect of the foregoing second feature, the SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe], or pharmaceutically acceptable salts thereof. In yet another aspect of the foregoing second feature, the SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In yet another aspect of the foregoing second feature, the SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 and the SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a third embodiment, the invention is directed to a method of decreasing the secretion of calcitonin from medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with an SSTR-1 agonist, wherein the medullary carcinoma cells are determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. In a first aspect of the third embodiment, the SSTR-2 agonist is a selective SSTR-2 agonist.
In a second aspect of the third embodiment, the SSTR-1 agonist or pharmaceutically acceptable salt thereof is an SSTR-1 selective agonist. The SSTR-1 selective agonist or pharmaceutically acceptable salt thereof may have a Ki value of less than 5 nM or even less than 1 nM.
In a third aspect of the third embodiment, exemplary SSTR-2 agonists useful to contact the medullary carcinoma cells to determine the change in calcitonin secretion include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In yet another feature, the preferred exemplary SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a fourth aspect of the third embodiment, exemplary SSTR-1 agonists include, but are not limited to, Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Aaeg-[D-Cys Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2, or Caeg-c[Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH, or pharmaceutically acceptable salts thereof. In one feature, the preferred SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the preferred SSTR-1 agonist is Aaeg-D-Cys-Pal-D-Trp-Lys-D-Cys)-T(Bzl)Tyr-NM or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-1 agonist is Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NE or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-1 agonist is Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-1 agonist is Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof.
In a fourth embodiment, the invention provides a method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with an SSTR-2 agonist either alone or in combination with an SSTR-3, SSTR-4 or SSTR-5 agonist, wherein the medullary carcinoma cells are determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. In a first aspect of the fourth embodiment, the SSTR-2, SSTR-3, SSTR-4 or SSTR-5 agonists or pharmaceutically acceptable salts thereof used to contact the medullary thyroid carcinoma cells are SSTR-2, SSTR-3, SSTR-4 or SSTR-5 selective agonists. In a second aspect of the fourth embodiment, the SSTR-2, SSTR-3, SSTR-4 or SSTR-5 agonists or pharmaceutically acceptable salts thereof used to contact the medullary thyroid carcinoma cells have Ki values of less than 5 nM. In a third aspect of the fourth embodiment, the SSTR-2, SSTR-3, SSTR-4 or SSTR-5 agonists or pharmaceutically acceptable salts thereof used to contact the medullary thyroid carcinoma cells have Ki values of less than 1 nM.
In a fourth aspect of the fourth embodiment, an SSTR-2 agonist or a pharmaceutically acceptable salt thereof is used to contact the contacted medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. Exemplary SSTR-2 agonists used to contact the contacted medullary thyroid carcinoma cells include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred SSTR-2 agonist used to contact the contacted medullary carcinoma cells is D-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In one feature, the preferred SSTR-2 agonist used to contact the contacted medullary carcinoma cells is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In one feature, the preferred SSTR-2 agonist used to contact the contacted medullary carcinoma cells is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist used to contact the contacted medullary carcinoma cells is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist used to contact the contacted medullary carcinoma cells is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist used to contact the contacted medullary carcinoma cells is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a fifth aspect of the fourth embodiment, a combination of an SSTR-2 agonist and an SSTR-3 agonist, or pharmaceutically acceptable salts thereof, is used to contact medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. In a sixth aspect of the fourth embodiment, a combination of an SSTR-2 agonist and an SSTR-4 agonist, or pharmaceutically acceptable salts thereof, is used to contact medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof.
In a seventh aspect of the fourth embodiment, a combination of an SSTR-2 agonist and an SSTR-5 agonist, or pharmaceutically acceptable salts thereof, is used to contact medullary thyroid carcinoma cells determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist or a pharmaceutically acceptable salt thereof. En one feature of the immediately foregoing seventh aspect, the SSTR-5 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a first feature of the seventh aspect of the fourth embodiment, the SSTR-5 agonist used to contact the medullary thyroid carcinoma cells in combination with an SSTR-2 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or a pharmaceutically acceptable salt thereof. Exemplary SSTR-2 agonists used to contact the medullary thyroid carcinoma cells in combination with the immediately foregoing SSTR-5 agonist include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-P-Trp-Lys-Abu-Cys]-Thr-NH, or pharmaceutically acceptable salts thereof. In one feature, the preferred SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In one feature, the preferred SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In another feature, the preferred SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a second feature of the seventh aspect of the fourth embodiment, the SSTR-5 agonist used to contact the medullary thyroid carcinoma cells in combination with an SSTR-2 agonist is D-Phe-[Cys-Tyr(1)-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. Exemplary SSTR-2 agonists used to contact the medullary thyroid carcinoma cells in combination with the immediately foregoing SSTR-5 agonist include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(ylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH or pharmaceutically acceptable salts thereof. In one feature, the preferred SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In one feature, the preferred SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In another feature, the preferred SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a fifth embodiment, the invention provides a method of decreasing the rate of proliferation of medullary thyroid carcinoma cells which comprises contacting medullary thyroid carcinoma cells with a somatostatin agonist which binds to both SSTR-2 and SSTR-5, or a pharmaceutically acceptable salt thereof. D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NH2, or a pharmaceutically acceptable salt thereof, is an exemplary SSTR-2 and SSTR-5 agonist compound.
In a sixth embodiment, the invention provides a method of treating medullary thyroid carcinoma which comprises administering to a patient in need thereof an effective amount of an SSTR-1 agonist, an SSTR-2 agonist, an SSTR-5 agonist, or a combination of one or more SSTR-1 agonist and one or more SSTR-2 agonist, wherein the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist. In a first aspect of the sixth embodiment, the SSTR-1, SSTR-2 or SSTR-5 agonists administered to the MTC patient are SSTR-1, SSTR-2 or SSTR-5 selective agonists or pharmaceutically acceptable salts thereof. In a second aspect of the sixth embodiment, the SSTR-1, SSTR-2 or SSTR-5 agonists or pharmaceutically acceptable salts thereof administered to the MTC patient have Ki values of less than 5 nM. In a third aspect of the sixth embodiment, the SSTR-1, SSTR-2, or SSTR-5 agonists or pharmaceutically acceptable salts thereof administered to the patient have Ki values of less than 1 nM.
In a fourth aspect of the sixth embodiment, an SSTR-1 agonist is administered to the MTC patient wherein the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist. In one feature of the immediately forgoing, the SSTR-1 agonist administered to the patient is an SSTR-1 selective agonist. Exemplary SSTR-1 agonists useful to practice the invention according to the fourth aspect of the sixth embodiment include, but are not limited to, Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2, or Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-1 agonist is Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-1 agonist is Taeg-c[P-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In yet another feature, the exemplary SSTR-1 agonist is Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof.
In a fifth aspect of the sixth embodiment, an SSTR-2 agonist is administered to the MTC patient wherein the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist. In one feature of the immediately forgoing, an SSTR-2 agonist is administered to the MTC patient wherein the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist. In another feature of the immediately foregoing, the SSTR-2 agonist administered to the patient is an SSTR-2 selective agonist. Exemplary SSTR-2 agonists useful to practice the invention according to the fifth aspect of the sixth embodiment include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 D-Phe-Cys-Phe-D-Trp-Lys Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-TyT-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-2 agonist administered to the patient is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-2 agonist administered to the patient is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In yet another feature, the preferred exemplary SSTR-2 agonist administered to the patient is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist administered to the patient is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist administered to the patient is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof. In yet another feature, the exemplary SSTR-2 agonist administered to the patient is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a sixth aspect of the sixth embodiment, an SSTR-5 agonist is administered to the MTC patient wherein the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist. In one feature of the immediately forgoing, the SSTR-5 agonist administered to the patient is an SSTR-5 selective agonist. Exemplary SSTR-5 agonists useful to practice the invention according to the sixth aspect of the sixth embodiment include, but are not limited to, D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-5 agonist is D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-5 agonist is D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NH or a pharmaceutically acceptable salt thereof.
In a seventh aspect of the sixth embodiment, a combination of an SSTR-1 agonist and an SSTR-2 agonist, or pharmaceutically acceptable salts thereof, is administered to the MTC patient wherein the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist. In one feature of the immediately foregoing, the SSTR-1 and the SSTR-2 agonists administered to the patient are SSTR-1 and SSTR-2 selective agonists or pharmaceutically acceptable salts thereof. In another feature of the seventh aspect of the sixth embodiment, exemplary SSTR-1 compounds in the combination include, but are not limited to Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2, or Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, or pharmaceutically acceptable salts thereof, and exemplary SSTR-2 compounds in the combination include, but are not limited to D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a first feature of the seventh aspect of the sixth embodiment the SSTR-1 agonist administered to the MTC patient in combination with an SSTR-2 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. Exemplary SSTR-2 agonists administered to the MTC pateint in combination with the immediately foregoing SSTR-1 agonist include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NHS D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2) c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a second feature of the seventh aspect of the sixth embodiment the SSTR-1 agonist administered to the MTC patient in combination with an SSTR-2 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH or a pharmaceutically acceptable salt thereof. Exemplary SSTR-2 agonists administered to the MTC pateint in combination with the immediately foregoing SSTR-1 agonist include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH. D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a seventh embodiment, the invention provides a method of treating medullary thyroid carcinoma which comprises administering to an MTC patient in need thereof an effective amount of an SSTR-1 agonist, wherein the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist. In a first aspect of the seventh embodiment, the SSTR-2 agonist is a selective agonist. In a second aspect of the seventh embodiment, the SSTR-1 agonist or pharmaceutically acceptable salt thereof is an SSTR-1 selective agonist. In one feature of the immediately foregoing second aspect, the SSTR-1 agonist or pharmaceutically acceptable salt thereof has a Ki value of less than 5 nM or even 1 nM.
In a third aspect of the seventh embodiment, exemplary SSTR-2 agonists include but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-2 agonist is D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-2 agonist is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol or a pharmaceutically acceptable salt thereof. In yet another feature, the preferred exemplary SSTR-2 agonist is Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is c[Tic-Tyr-D-Trp-Lys-Abu-Phe] or pharmaceutically acceptable salts thereof. In another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-2 agonist is 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 or a pharmaceutically acceptable salt thereof.
In a fourth aspect of the seventh embodiment, exemplary SSTR-1 agonists administered to the MTC patient include, but are not limited to Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2, or Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, or pharmaceutically acceptable salts thereof. In one feature, the preferred exemplary SSTR-1 agonist is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the preferred exemplary SSTR-1 agonist is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In yet another feature, the preferred exemplary SSTR-1 agonist is Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In another feature, the exemplary SSTR-1 agonist is Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof. In yet another feature, the exemplary SSTR-1 agonist is Caeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof.
In an eighth embodiment, the invention provides a pharmaceutical composition comprising an effective amount of an SSTR-1 agonist or a pharmaceutically acceptable salt thereof and an SSTR-2 agonist or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent. In a first aspect of the eighth embodiment, the SSTR-1 agonist is a selective SSTR-1 agonist and the SSTR-2 agonist is a selective SSTR-2 agonist. In a second aspect of the eighth embodiment, exemplary SSTR-1 agonists in the pharmaceutical composition include, but are not limited to, Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, Taeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Ser(Bzl)-Tyr-NE, or Caeg-c[p-Cys-Phe-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH, and exemplary SSTR-2 agonists in the pharmaceutical composition include, but are not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperazinylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperazine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a preferred aspect of the eighth embodiment, the SSTR-1 agonist in the pharmaceutical composition is Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-N or a pharmaceutically acceptable salt thereof in combination with SSTR-2 agonists such as, but not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In another preferred aspect of the eighth embodiment, the SSTR-1 agonist in the pharmaceutical composition is Aaeg-[D-Cys-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 or a pharmaceutically acceptable salt thereof in combination with SSTR-2 agonists such as, but not limited to, D-β-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, Dop2-D-Lys(Dop2)-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, c[Tic-Tyr-D-Trp-Lys-Abu-Phe], 4-(2-Hydroxyethyl)-1-piperas(inylacetyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or 4-(2-Hydroxyethyl)-1-piperas(ine-2-ethanesulfonyl-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2, or pharmaceutically acceptable salts thereof.
In a ninth embodiment, the pharmaceutical composition of the immediately foregoing eighth embodiment is used to treat medullary thyroid carcinoma. In a first aspect of the ninth embodiment, the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist. In a second aspect of the ninth embodiment, the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist.
In a tenth embodiment, the invention provides a pharmaceutical composition comprising an effective amount of an SSTR-1 and SSTR-2 agonist or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent. In a further aspect of the tenth embodiment, the SSTR-1 and SSTR-2 agonist is selective for both the somatostatin subtype-1 receptor and the somatostatin subtype-2 receptor. In yet a further aspect of the tenth embodiment, the SSTR-1 and SSTR-2 agonist is selective for both the somatostatin subtype-1 receptor and the somatostatin subtype-2 receptor.
In an eleventh embodiment, the pharmaceutical composition of the immediately foregoing tenth embodiment is used to treat medullary thyroid carcinoma. In a first aspect of the ninth embodiment, the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of greater than about 15% in response to administration of an SSTR-2 agonist in a second aspect of the ninth embodiment, the medullary carcinoma is determined to exhibit a decrease in calcitonin secretion of less than about 15% in response to administration of an SSTR-2 agonist.
In a twelfth embodiment, the invention provides a method according to any of the foregoing embodiments, wherein at least one SSTR-1 agonist and SSTR-2 agonist, or pharmaceutically acceptable salts thereof, comprise a Tyr(1) residue, wherein the iodine atom of the Tyr(1) residue comprises a radioactive iodine isotope. In a first aspect of the twelfth embodiment, the medullary thyroid carcinoma cells have formed metastases outside the thyroid. In a second aspect of the twelfth embodiment, the radioactive iodine is 125I, 127I or 131I. I a third aspect of the twelfth embodiment, the metastases are present in the lymph, the lung, the liver, the brain, or in bone. In a first feature of the twelfth embodiment or of the first, second or third aspects of the twelfth embodiment, the preferred methods are according to the second, third, fourth, sixth and seventh embodiments of the invention as discussed above.
Qualitative RT-PCR was performed on 36 MTC samples, showing the presence or absence of the expression of each SSTR The bars indicate the percentage of positive samples.
Primary cultured cells from 18 selected MTC samples were incubated in ninety six well plates for six hours with each SSTR agonist at 10−8 M. Control cells were treated with vehicle solution. Calcitonin levels in the conditioned medium were measured by immunoradiometric assay (IRMA) among eight replicates in the conditioned medium from treated and untreated (control) primary cultured cells. As described herein, samples were divided according to calcitonin secretion inhibition after treatment with lanreotide in group A (responders, black bars) and group B (non responders, white bars). Data are expressed as the mean±SE percent hormone secretion inhibition vs. untreated control cells. *P<0.05 and **P<0.01 vs. control.
The compounds represented in
Compound #1: Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH
Compound #2: c[Tic-Tyr-D-Trp-Lys-Abu-Phe]
Compound #3: D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2
Compound #4: D-Phe-[Cys-Tyr(1)-D-Trp-Lys-Val-Cys]-Thr-NH
Primary cultured cells from 18 selected MTC samples were incubated in ninety six well plates for forty eight hours with each SSTR agonist at 10−8 M. Control cells were treated with vehicle solution. Cell viability was measured by a colorimetric method among eight replicates from treated and untreated (control) primary cultured cells. As described herein, samples were divided according to calcitonin secretion inhibition after treatment with lanreotide in group A (responders, black bars) and group B (non responders, white bars). Data are expressed as the mean±SE percent cell viability reduction vs. untreated control cells. *P<0.05, and **P<0.01 vs. control.
The compounds represented in
Compound #1: Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2
Compound #2: c[Tic-Tyr-D-Trp-Lys-Abu-Phe]
Compound #3: D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH
Compound #4: D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NE
It is believed that one skilled in the art can, based on the description herein, utilis(e the present invention to its fullest extent The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents and other references mentioned herein are incorporated by reference, each in its entirety.
Various somatostatin receptors have been isolated, e.g., SSTR-1, SSTR-2, SSTR-3, SSTR-4, and SSTR-5. Thus, a somatostatin agonist may be one or more of an SSTR-1 agonist, SSTR-2 agonist, SSTR-3 agonist, SSTR-4 agonist or a SSTR-5 agonist.
What is meant by an SSTR-1 receptor agonist (i.e., SSTR-1 agonist) is a compound which has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nM or less than 1 nM) for SSTR-1 (e.g., as defined by the receptor binding assay in U.S. Pat. No. 7,084,117 incorporated herein by reference in its entirety). What is meant by an SSTR-1 receptor selective agonist is an SSTR-1 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-1 than for another receptor, i.e., SSTR-2, SSTR-3, SSTR-4 or SSTR-5.
What is meant by an SSTR-2 receptor agonist is a somatostatin agonist which has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nM or less than 1 nM) for SSTR-2 (e.g., as defined by the receptor binding assay in U.S. Pat. No. 7,084,117 incorporated herein by reference in its entirety). What is meant by an SSTR-2 receptor selective agonist is an SSTR-2 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-2 than for any other somatostatin receptor i.e., SSTR-1, SSTR-3, SSTR-4 or SSTR-5.
What is meant by an SSTR-3 receptor agonist is a somatostatin agonist which has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nM or less than 1 nM) for SSTR-3 (e.g., as defined by the receptor binding assay in U.S. Pat. No. 7,084,117 incorporated herein by reference in its entirety). What is meant by an SSTR-3 receptor selective agonist is an SSTR-3 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-3 than for any other somatostatin receptor i.e., SSTR-1, SSTR-2, SSTR-4 or SSTR-5.
What is meant by an SSTR-4 receptor agonist is a somatostatin agonist which has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nM or less than 1 nM) for SSTR-4 (e.g., as defined by the receptor binding assay in U.S. Pat. No. 7,084,117 incorporated herein by reference in its entirety). What is meant by an SSTR-4 receptor selective agonist is an SSTR-4 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-4 than for any other somatostatin receptor i.e., SSTR-1, SSTR-2, SSTR-3 or SSTR-5.
What is meant by an SSTR-5 receptor agonist is a somatostatin agonist which has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nM or less than 1 nM) for SSTR-5 (e.g., as defined by the receptor binding assay in U.S. Pat. No. 7,084,117 incorporated herein by reference in its entirety). What is meant by an SSTR-5 receptor selective agonist is an SSTR-5 receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-5 than for any other somatostatin receptor i.e., SSTR-1, SSTR-2, SSTR-3 or SSTR-4.
Some somatostatin agonist compounds exhibit high binding affinities for two, or even three, somatostatin receptors as compared to other somatostatin receptors. Such somatostatin agonists are also classified as a somatostatin agonist wherein the compound has a high binding affinity (e.g., Ki of less than 100 nM or preferably less than 10 nM or less than 1 nM) for two (or three) different somatostatin receptors (e.g., as defined by the receptor binding assay in U.S. Pat. No. 7,084,117 incorporated herein by reference in its entirety). Thus, what is meant by an SSTR-5 and SSTR-2 receptor agonist is a receptor agonist which has a higher binding affinity (i.e., lower Ki) for SSTR-5 and for SSTR-2 than for other somatostatin receptors, i.e., SSTR-1, SSTR-3 or SSTR4.
In one embodiment, the SSTR-1 agonist is also an SSTR-1 selective agonist and the SSTR-2 agonist is also an SSTR-2 selective agonist
Examples of SSTR-1 agonists which may be used to practice the present invention includes, but is not limited to:
Examples of SSTR-2 agonists which may be used to practice the present invention include, but are not limited to:
Examples of SSTR-5 agonists which may be used to practice the present invention include, but are not limited to:
An example of an agonist binding to two different SSTR receptors, in this case, SSTR-5 and SSTR-2, which may be used to practice the present invention includes, but is not limited to, D-Phe-[Cys-Tyr(1)-D-Trp-Lys-Val-Cys]-Thr-NH.
Further examples of somatostatin agonists are those covered by formulae or those specifically recited in the publications set forth below, all of which are hereby incorporated by reference.
Note that for all somatostatin agonists described herein, each amino acid residue represents the structure of —NH—C[R)H—CO—, in which R is the side chain (e.g., CH3 for Ala). Lines between amino acid residues represent peptide bonds which join the amino acids. Also, where the amino acid residue is optically active, it is the L-form configuration that is intended unless D-form is expressly designated. For clarity, disulfide bonds (e.g., disulfide bridge) which exist between two free thiols of Cys residues are not shown. Abbreviations of the common amino acids are in accordance with IUPAC-IUB recommendations.
Some of the compounds of the instant invention can have at least one asymmetric center. Additional asymmetric centers may be present on the molecule depending upon the nature of the various substituents on the molecule. Each such asymmetric center will produce two optical isomers and it is intended that all such optical isomers, as separated, pure or partially purified optical isomers, racemic mixtures or diastereomeric mixtures thereof, are included within the scope of the instant invention.
The compounds of the instant invention generally can be isolated in the form of their pharmaceutically acceptable acid addition salts, such as the salts derived from using inorganic and organic acids. Examples of such acids are hydrochloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, D-tartaric, L-tartaric, malonic, methane sulfonic and the like. In addition, certain compounds containing an acidic function such as a carboxy can be isolated in the form of their inorganic salt in which the counter-ion can be selected from sodium, potassium, lithium, calcium, magnesium and the like, as well as from organic bases.
The pharmaceutically acceptable salts can be formed by taking about 1 equivalent of an SSTR-2 agonist, e.g., c[Tic-Tyr-D-Trp-Lys-Abu-Phe], and contacting it with about 1 equivalent or more of the appropriate corresponding acid of the salt which is desired. Work-up and isolation of the resulting salt is well-known to those of ordinary skill in the art.
The compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration. Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one SSTR-1 agonist and at least one SSTR-2 agonist in association with a pharmaceutically acceptable carrier or a combination of two pharmaceutical compositions wherein the first is comprised of an active ingredient of at least one SSTR-1 agonist in association with a pharmaceutically acceptable carrier and the second is comprised of an active ingredient of at least one SSTR-2 agonist in association with a pharmaceutically acceptable carrier.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilis(ed by, for example, filtration through a bacteria-retaining filter, by incorporating sterilis(ing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.
Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
To best treat those patients, the time to resolution of the symptoms should be rapid and coincide with the suppression of circulating calcitonin levels. Treatment of symptomatic, refractory would be a life time treatment in a patient population with a reasonable life expectancy.
In general, an effective dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment, all of which are within the realm of knowledge of one of ordinary skill in the art. Generally, dosage levels of between 0.0001 to 100 mg/kg of body weight daily are administered to humans and other animals, e.g., mammals.
A preferred dosage range is 0.01 to 10.0 mg/kg of body weight daily, which can be administered as a single dose or divided into multiple doses.
The methods for synthesis(ing somatostatin agonists are well documented and are within the ability of a person of ordinary skill in the art.
Synthesis of short amino acid sequences is well established in the peptide art. For example, synthesis of H-D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2, described above, can be achieved by following the protocol set forth in Example I of European Patent Application 0 395 417 A1 (incorporated herein by reference in its entirety). The synthesis of somatostatin agonists with a substituted N-terminus can be achieved, for example, by following the protocol set forth in WO 88/02756 (incorporated herein by reference in its entirety), European Patent Application No. 0 329 295(incorporated herein by reference in its entirety), and PCT Publication No. WO 94/04752 (incorporated herein by reference in its entirety).
Eighteen medullary thyroid carcinoma samples, obtained from donors averaging 55.4 years in age (±3 years with median 54 years old), were selected on the basis of verification of calcitonin, CgA, SSTR-1, SSTR-2 and SSTR-5 mRNA expression by RT-PCR analysis of an initial group of thirty-six samples obtained from patients diagnosed with medullary thyroid carcinoma which were surgically removed. All surgeries took place at the Section of Endocrinology and at the Section of General Surgery of the University of Ferrara, Ferrara, Italy and at the Department of General Surgery III of the University of Padua, Padua, Italy between 2003 and 2005. Table I shows patients' characteristics and pre-operative hormonal values.
All patients (8 males and 10 females; age=50.3±4.6 years old) underwent a total thyroidectomy with central neck lymph node clearance after histological and immunohistochemical diagnosis of medullary thyroid carcinoma. Tissue from the primary tumour was obtained from thirteen patients of the selected patient population while tissue from the metastatic lymph nodes of five different members of the test population was also procured.
Tissue samples were surgically removed in accordance with the guidelines of the local comnmittees on human research. Thyroid tissue samples were obtained under sterile conditions and immediately minced in RPMI 1640 tissue culture medium. Tissues were incubated with 0.25% trypsin overnight at 4° C. and then dissociated using 0.35% collagenase (Sigma, Milano, ITALY) and 1% trypsin at 37° C. for sixty minutes. Cell suspensions were filtered through double layers of gaus(e and washed twice with serum-free F-12 Ham's Nutrient Modified Medium (F-12; Euroclone Ltd, Wetherby, UNITED KINGDOM). Tumor cells were re-suspended in F-12 with 10% fetal bovine serum and antibiotics, seeded in ninety-six well culture plates (˜2×104 cells/well) and incubated at 37° C. in a humidified atmosphere of 5% CO2 and 95% air according to known procedures (Zatelli, M. C. et al., Endocrinology, 2005, 146:2692-8). Approximately twenty-four hours later, the prepared cells were treated with the selected somatostatin agonist and were monitored for hormone secretion and overall viability. Informed consent of the patients was obtained for disclosing clinical investigation and performing the in vitro study.
In order to demonstrate the origin of the samples from parafollicular C-cells, RT-PCR analysis for calcitonin expression was performed on each specimen. Further expression analysis for CgA, SSTR-1, SSTR-2, SSTR-3, SSTR-4 and SSTR-5 was performed with only calcitonin expressing tissues as previously described in the art (Zatelli, M. C. et al., J. Clin. Endocrinol. Metab., 2005, 90:2104-9). Fros(en tissues were disrupted and total RNA from the pulveris(ed tumours was isolated with TRIZOL® reagent (Invitrogen, Milano, ITALY) according to the manufacturer's protocol.
To prevent DNA contamination, the RNA was treated with RNase-free deoxyribonuclease (Promega; Milano, ITALY). Using a first strand complementary DNA (cDNA) synthesis kit (SuperScript Preamplification System for First Strand cDNA Synthesis®, Invitrogen, Milano, ITALY), 1 μg of total RNA was reverse transcribed with random hexamers according to the manufacturer's protocol. The reverse transcription (RT) reaction was carried out in a GeneAmp 9700 PCR System® (Applera Italia, Mons(a, ITALY) using previously known protocols (Zatelli, M. C. et al., Horm. Metab. Res., 2003, 35:349-54).
The cDNA (1 μl of RT reaction) was then amplified by PCR with 1 U Taq DNA polymerase (Invitrogen, Milano, ITALY) under conditions recommended by the supplier. Integrity of the cDNA was tested by demonstrating the presence of a GAPDH signal. PCR reactions were carried out using the oligonucleotide primers as detailed in Table 2 and as previously described (Zatelli, M. C. et al., Horm. Metab. Res., 2002, 34:229-33; Zatelli, M. C. et al., J. Clin. Endocrinol. Metab., 2004, 89:5181-8; Zatelli, M. C. et al., J. Clin. Endocrinol. Metab., 2003, 88:2797-802).
2D) Quantitative PCR for SSTR-1. SSTR-2 and SSTR-5 mRNA
Using the human endogenous control plate (Applera Italia, Mons(a, ITALY) according to the manufacturer's instructions, an endogenous control gene was selected. Total RNA was isolated as described above from three medullary thyroid carcinoma samples and from a pool of three samples of normal thyroid tissue deriving from the same patient. The candidate genes included 18S rRNA, acidic ribosomal protein, β-actin, cyclophilin, GAPDH, phosphoglycerokinase, P2-microglobulin, human β-glucuronidase (huGUS), hypoxanthine ribosyl transferase, transcription factor IID TATA-binding protein, and transferrin receptor. With the exception of 18S rRNA, the primers and the probes of the candidate genes recognis(ed only cDNA. All measurements were performed in duplicate experiments. The results are expressed as “threshold cycle value (ACT)” representing the difference between the threshold cycle obtained of the normal thyroid pool and the threshold cycle recorded for the samples for each housekeeping gene. The normal thyroid pool value serves as a baseline for the assays and is considered as s(ero.
Quantitative PCR for SSTR-1, SSTR-2 and SSTR-5 was performed using procedures previously disclosed (Zatelli, M. C. et al., Endocrinology, 2005, 146:2692-8; Zatelli, M. C. et al., J. Clin. Endocrinol. Metab., 2003, 88:2797-802) with primers and probes designed using Primer Express Software 0 (Applera Italia, Mons(a, ITALY). TaqMan® probes (Applera Italia, Mons(a, ITALY) labelled with a fluorescent dye (6-carboxy-fluorescein, FAM) and a quencher dye (6-carboxy-tetramethyl rhodamine, TAMRA) were used. Pre-Developed TaqMan® Assay Reagents for huGUS (20×) were used to assess retrotranscription efficiency (Applera Italia, Mons(a, ITALY).
Serial dilutions of the single stranded SSTR-1, SSTR-2 and SSTR-5 sense oligonucleotide amplicons (from 109 to 102 molecules) were carried out in triplicate. The log copy numbers of unknown samples were calculated from the regression line according to the equation: log N=(Ct−q)/m, where Ct is the threshold cycle, q is the y-intercept, and m is the slope of the standard curve line. All Quantitative PCR reactions were performed, recorded and analysed using the ABI 7700 Prism Sequence Detection System® (Applera Italia, Mons(a, ITALY). Slopes for all assays reported were −3.3±0.1.
All samples were carried out in triplicate (100 ng of reverse transcribed total RNA per well) and repeated at least twice. No Template Control and RT—controls were run in each experiment.
A cut-off of 3×103 mRNA copies/μg total RNA was established as the threshold for Real Time PCR to exclude the detection of transcripts due to illegitimate transcription, as previously suggested (Korbonits, M. et al., J. Clin. Endocrinol. Metab., 2001, 86:881-7; and Chelly, J. et al., Proc. Natl. Acad. Sci. USA, 2001, 86:2617-21).
Somatostatin analogs (IPSEN (Biomeasure, Inc.), Milford, Mass., UNITED STATES OF AMERICA) used in this study and their respective affinities to the different SSTR are listed below in Table 3:
The effects of the tested somatostatin analogs on medullary thyroid carcinoma primary culture cell viability were assessed with a Cell Proliferation Kit I MTT® (Roche Diagnostics GmbH, Mannheim, GERMANY) which employs a colorimetric method for determining the number of viable cells in proliferation assays. Primary cultured cells were plated in ninety-six multiwell plates (2×104 cells/well) and incubated for forty-eight hours in a medium supplemented with 10% fetal bovine serum in the presence or absence of each somatostatin analog at 10−8M. Treatments were renewed after the first twenty-four hours of incubation. The absorbance at 560 nm was then recorded using microplate reader (Victor, Perkin Elmer, Mons(a, ITALY). Results (absorbance at 560 nm) were obtained by determining the mean value of at least six experiments in eight replicates using known procedures previously described (Zatelli, M. C. et al., Biochem. Biophys. Res. Commun., 2002, 297:828-34).
Calcitonin levels were measured in a conditioned medium from treated and untreated primary cultured cells with the calcitonin-U.S.-IRMA® kit (Biosource Europe, Nivelles, BELGIUM) after a six hour treatment with 1046 M of the selected somatostatin analogs. The intra- and inter-assay variation coefficients were 1.9-2.7% and 1.9-3.3%, respectively. The detection limit was 0.8 pg/ml. Hormone assays were performed in duplicate of conditioned medium from treated cells. Results were obtained by determining the mean value among eight replicates. Primary cultures were considered as “responders” when a significant reduction >15% in calcitonin secretion was recorded under treatment with lanreotide.
The effects of the selected somatostatin analogs on CgA secretion were analysed by measuring human CgA immunoreactivity in the culture medium from primary cultured cells incubated for six hours with or without 10−8M each somatostatin analog using an ELISA kit (Dako, Milano, ITALY), according to the process previously described (Zatelli, M. C. et al., Horm. Metab. Res., 2003, 35:349-54). The detection limit was 2.0 U/L, with intra- and inter-assay coefficients of variation of 5.8% and 8.6%, respectively. Hormone assays were performed in duplicate. Results were obtained by determining the mean value among eight replicates.
Data are expressed as the mean±standard error (SE). A preliminary analysis was carried out to determine whether the datasets conformed to a normal distribution and a computation of homogeneity of variance was performed using Bartlett's test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P<0.05), then either Student's paired or unpaired t test was used to evaluate the individual differences between means. To measure the strength of association between pairs of variables without specifying dependencies Spearman order correlations were run. A P<0.05 was considered significant in all tests.
Thirty-six surgically removed medullary thyroid carcinoma samples underwent RT-PCR analysis for calcitonin, CgA, and SSTR1-5 mRNA expression and eighteen tumours (50%) were selected on the basis of the expression of calcitonin, CgA, SSTR-1, SSTR-2 and SSTR-5 mRNAs (see Table 4; see also
The absolute SSTR m-RNA levels were also investigated in the selected samples. By employing the human endogenous control plate, it was learned that the best endogenous control gene, expressed at nearly constant levels in all samples examined, was the huGUS. The assays produced Δcalcitonin values for huGUS slightly different from s(ero in all examined samples indicating a fairly stable level of gene expression in the samples as compared to the normal thyroid pool. Quantitative PCR showed SSTR-1 mRNA levels of 6.8 105 copies/μg total RNA, SSTR-2 of 7.3×107 molecules/μg total RNA, and SSTR-5 of 8.5×104 copies/μg total RNA. SSTR mRNA expression levels are displayed in Tables 5 and Tables 6.
§Responder and
†non responder MTC primary cultures
§Responder and
†non responder MTC primary cultures
In order to investigate the effects of somatostatin analogs on hormone secretion, calcitonin and CgA levels were measured in conditioned media collected from primary cultures derived from the selected eighteen medullary thyroid carcinoma treated with or without each somatostatin analog at 10−8M for six hours. As indicated in Table 4, measurable amounts of calcitonin and CgA were detected in the culture medium from all cultured tumour tissues. Percent calcitonin secretion reduction compared with control in each primary culture under treatment with each somatostatin analog is displayed in Table 5.
The primary cultures were then divided in two groups according to the extent of calcitonin secretion inhibition recorded after treatment with D-β-Nal-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2 (sold as lanreotide). Cultures responding to lanreotide with a calcitonin reduction of 15% or greater when compared to untreated cells were considered “responders”. According to this criterion, nine cultures were considered as “responders” (group A: primary cultures #3, #5, #6, #7, #11, #12, #13, #14, #15, #16) and nine as “non responders” (group B: primary cultures #1, #2, #4, #8, #9, #10, #17, #18). SSTR mRNA levels did not significantly differ between the two groups. Group A displayed SSTR-1 mRNA levels of 9.1×105 copies/μg total RNA, SSTR-2 of 9.7×107 copies/μg total RNA and SSTR-5 of 1.5×105 copies/μg total RNA. Group B displayed SSTR-1 mRNA levels of 4.6×105 copies/μg total RNA, SSTR-2 of 4.9×107 copies/μg total RNA and SSTR-5 of 1.7×104 copies/μg total RNA. On the other hand, mean pre-operative calcitonin plasma levels were significantly lower in patients belonging to group A than in patients belonging to group B (158±105 pg/ml vs. 2383±963 pg/ml; P<0.01).
In group A, calcitonin secretion was reduced by 27% after treatment with lanreotide (
In group B, which did not respond to lanreotide, it was discovered that calcitonin secretion was not reduced by treatment with somatostatin analogs interacting with SSTR-2, SSTR-5 or both (c[Tic-Tyr-D-Trp-Lys-Abu-Phe], D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2, D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NH2). On the other hand, the SSTR-1 selective agonist, Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2, significantly reduced calcitonin secretion (−13%; P<0.05) (
With regard to CgA secretion, the somatostatin analogs did not significantly affect CgA secretion in medullary thyroid carcinoma primary cultures, in either group A or group B, except for medullary thyroid carcinoma #8. In this sample, belonging to the “non responder” group expressing all SSTR receptors, a significant reduction in CgA secretion after treatment with each SSTR selective agonist alone or in combination (ranging from −35% to −67% CgA secretion vs. control; P<0.05) was noted.
In order to investigate the effects of somatostatin analogs on medullary thyroid carcinoma cell viability, an MTT assay was performed in primary cultures derived from the eighteen selected medullary thyroid carcinoma samples, treated with or without each somatostatin analog at 10M for forty-eight hours. Percent cell viability reduction compared to a control in each primary culture under treatment with each somatostatin analog is reported in Table 6.
In general, cellular viability of cells from primary MTC cultures designated as responsive to lanreotide (Group A) was not reduced by treatment with any particular somatostatin analog. In contrast, cellular proliferation of cells from primary MTC cultures designated as non-responsive to lanreotide (Group B) was reduced following treatment with lanreotide (−14%; P<0.01), the SSTR-2 selective agonist c[Tic-Tyr-D-Trp-Lys-Abu-Phe] (−13%; P<0.05) and the dual SSTR-2/SSTR-5 selective agonist, D-Phe-[Cys-Tyr(I)-D-Trp-Lys-Val-Cys]-Thr-NH2 (−13%; P<0.05). Medullary thyroid carcinoma cell viability was neither affected by treatment with the SSTR-1 selective agonist, Caeg-c[D-Cys-3-Pal-D-Trp-Lys-D-Cys]-Thr(Bzl)-Tyr-NH2 nor the SSTR-5 selective agonist, D-Phe-Phe-Trp-D-Trp-Lys-Thr-Phe-Thr-NH2. Moreover, the combination of the SSTR-1 and the SSTR-2 selective agonists did not compromise primary culture cell viability.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/007339 | 3/23/2007 | WO | 00 | 9/23/2008 |
