Methods and compositions for forming copper-containing complexes and uses thereof
阅读说明：本技术 用于形成含铜络合物的方法和组合物及其用途 (Methods and compositions for forming copper-containing complexes and uses thereof ) 是由 尼古拉斯·汤克斯 纳瓦索纳·克里希南 安德烈亚斯·格里尔 霍华德·萨尔德 于 2018-11-06 设计创作，主要内容包括：提供了形成含铜络合物的方法,包括使含铜样品与式I化合物接触：其中R是-OH或-O-CH<Sub>3</Sub>。还提供了抑制样品中激酶的酶活性的方法,包括使样品与式I化合物接触。还提供了向受试者施用包括任选地与铜络合的式I化合物的药物组合物的方法。还提供了包括与式I化合物络合的铜的药物组合物。<Image he="229" wi="700" file="DDA0002568980410000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(There is provided a method of forming a copper-containing complex comprising contacting a copper-containing sample with a compound of formula I: wherein R is-OH or-O-CH 3 . Also provided are methods of inhibiting the enzymatic activity of a kinase in a sample comprising contacting the sample with a compound of formula I. Also provided is the administration of a medicament comprising a compound of formula I, optionally complexed with copper, to a subjectA method of making the composition. Also provided are pharmaceutical compositions comprising copper complexed with a compound of formula I.)
1. A method of forming a copper-containing complex comprising contacting a copper-containing sample with a compound of formula I:
wherein R is-OH or-O-CH3。
2. The method of claim 1, wherein the copper-containing sample is a non-biological solution or suspension.
3. The method of claim 1, wherein the copper-containing sample is a biological solution, suspension, tissue, or organism.
4. The method of claim 3, wherein contacting a sample comprises administering the compound to a subject.
5. The method of claim 4, wherein administering the compound reduces the cytotoxic effect of copper.
6. The method of claim 5, wherein the subject is a human diagnosed with a disorder associated with an elevated physiological level of copper.
7. The method of claim 6, wherein the disorder is Wilson's disease.
8. The method of claim 1, comprising inhibiting a catalytic activity of an enzyme by forming the copper-containing complex, and the enzyme is selected from the group consisting of: pyruvate kinase m (pkm), mitochondrial adenylate kinase 2(AK2), creatine kinase B (ckb), p21 activated kinase (PAK), TP53 regulated kinase (TP53RK), phosphoglycerate kinase 1(PGK1), pyridoxal kinase (PDXK), U-type mitochondrial creatine kinase (CKMT1B), mitogen-activated protein kinase (MEK), tyrosine kinase CSK, protein tyrosine phosphatase 1B (PTP1B), and any combination of two or more of the foregoing.
9. A method of inhibiting the enzymatic activity of a kinase in a sample comprising contacting the sample with a compound of formula I:
wherein R is-OH or-O-CH3And an
Wherein the kinase is selected from the group consisting of: pyruvate kinase m (pkm), mitochondrial adenylate kinase 2(AK2), creatine kinase b (ckb), p21 activated kinase (PAK), TP53 regulated kinase (TP53RK), phosphoglycerate kinase 1(PGK1), pyridoxal kinase (PDXK), U-type mitochondrial creatine kinase (CKMT1B), mitogen-activated protein kinase (MEK), tyrosine kinase CSK, and any combination of two or more of the foregoing.
10. The method of claim 9, wherein the sample comprises a subject in need of drug treatment and contacting a sample comprises administering the compound to the subject.
11. The method of claim 10, wherein the subject has gastric cancer.
12. The method of claim 10, wherein the subject has HER2 negative breast cancer.
13. The method of claim 12, wherein the HER2 negative cancer is an estrogen receptor negative cancer, a progestin receptor negative cancer, or a triple negative breast cancer.
14. The method of claim 10, wherein the subject has triple negative breast cancer.
15. The method according to any one of claims 11, 12, 13 or 14, further comprising administering AZD6244 to the subject.
16. The method according to claim 15, wherein administration of the compound and AZD6244 to the subject has a therapeutic effect, and the therapeutic effect is greater than the therapeutic effect of administration of the compound to the subject without AZD6244 and the therapeutic effect of administration of AZD6244 to the subject without the compound.
17. The method of claim 16, wherein the therapeutic effect comprises inhibiting tumor growth, inhibiting metastasis of cancer cells, stimulating death of cancer cells, or any combination of two or more of the foregoing.
18. The method according to any one of claims 11, 12, 13 or 14 wherein the subject was previously administered AZD 6244.
19. The method of claim 18, wherein contacting the sample with the compound comprises inhibiting tumor growth, inhibiting metastasis of a cancer cell, stimulating death of a cancer cell, or any combination of two or more of the foregoing.
20. A method of administering to a subject a pharmaceutical composition comprising copper complexed with a compound of formula I:
wherein R is-OH or-O-CH3。
21. The method of claim 20, wherein administering the pharmaceutical composition comprises decreasing the body mass index of the subject.
22. The method of claim 20, wherein administering the pharmaceutical composition comprises enhancing a physiological response to one or more hormones, and the one or more hormones is insulin, leptin, or both.
23. The method of claim 20, wherein the subject has cancer and the cancer is gastric cancer or HER2 negative breast cancer and administering the pharmaceutical composition comprises inhibiting tumor growth, inhibiting cancer cell metastasis, stimulating cancer cell death, or any combination of two or more of the foregoing.
24. The method of claim 23, wherein the cancer is HER2 negative breast cancer, wherein the HER2 negative breast cancer is estrogen receptor negative, progestin receptor negative, or triple negative breast cancer.
25. The method of claim 24, wherein the cancer is triple negative breast cancer.
26. The method according to any one of claims 23, 24 or 25, further comprising administering AZD6244 to the subject.
27. The method according to claim 26, wherein administration of the compound and AZD6244 to the subject has a therapeutic effect, and the therapeutic effect is greater than the therapeutic effect of administration of the compound to the subject without AZD6244 and the therapeutic effect of administration of AZD6244 to the subject without the compound.
28. The method of claim 27, wherein the therapeutic effect comprises inhibiting tumor growth, inhibiting metastasis of cancer cells, stimulating death of cancer cells, or any combination of two or more of the foregoing.
29. The method according to any one of claims 23, 24 or 25 wherein the subject was previously administered AZD 6244.
30. The method of claim 29, wherein administering the pharmaceutical composition comprises inhibiting tumor growth, inhibiting metastasis of cancer cells, stimulating death of cancer cells, or any combination of two or more of the foregoing.
31. The method of claim 20, wherein administering the compound comprises inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B) in the subject.
32. A pharmaceutical composition comprising copper complexed with a compound of formula I:
wherein R is-OH or-O-CH3。
33. The pharmaceutical composition of claim 32, further comprising a pharmaceutically acceptable excipient.
Technical Field
The subject matter disclosed herein relates to compounds for complexing with copper ions, as well as compounds that complex with copper ions and uses thereof. More specifically, 3- (substituted amino) -7-hydroxy-cholanic acid derivatives that form complexes with copper, and their use as copper chelator/enzyme activity inhibitors and disease treatments, are disclosed.
Background
Dysregulation of signal transduction pathways, and the consequent disruption of the normal pattern of protein phosphorylation, has been implicated in the etiology of a number of major human diseases, including diabetes, obesity, and cancer. The ability to target such signal transduction pathways selectively holds tremendous therapeutic potential. Protein kinases have become major drug targets in the past decade. In particular, several drugs directed against protein kinases have had a major impact on the treatment of various cancers. However, the focus on kinases for drug development has met several challenges, including intrinsic and acquired resistance to such therapies. Thus, other targets and methods are needed. In this context, it is important to remember that protein phosphorylation is a reversible process in which the synergistic and competitive activities of kinases and phosphatases are important for determining the outcome of signal transduction. Protein Tyrosine Phosphatases (PTPs) represent a large class of proteins that act synergistically with kinases to control cell signal transduction and are also implicated in the etiology of several human diseases. However, PTPs are still underutilized as therapeutic targets. It is particularly important to develop treatments for diabetes, obesity and cancer by targeting the activity of phosphatases and kinases.
In addition, by controlling the activity of kinases such as MEK, it has been shown that copper is involved in the regulation of signal transduction, linking copper to the control of cell growth, and its destruction in tumorigenesis and metastasis. Physiological levels of copper are under complex homeostatic control, including control of influx and efflux transporters, and specialized chaperones that deliver metals to their site of action. Disruption of these homeostatic mechanisms is associated with a variety of disease states. The ATP7B mutation, which plays a role in copper excretion, leads to metal accumulation, resulting in Wilson's disease, a severe autosomal recessive genetic disorder. In particular, the physiological burden of the disease is felt in the liver, as this tissue expresses high levels of ATP 7B. It begins at a pre-symptomatic period during which copper accumulates in the liver. Various liver problems are encountered, ranging from hepatomegaly to hepatitis and cirrhosis, and even acute liver failure. As the disease progresses, it leads to the development of neuropsychiatric symptoms, including speech and cognitive disorders, particularly tremor and dystonia, as well as ataxia and parkinson's disease. In addition, at some point in the course of the disease, Wilson's patients develop mental problems, including personality changes, antisocial behavior, anxiety, and depression. Current treatment strategies rely on chelators that act as "decoppering" agents, with the aim of reducing the levels of metals and attempting to restore normal homeostasis. Unfortunately, the most commonly used drugs, penicillamine and trientine, are associated with severe adverse effects. Thus, there is a need for new potent and specific copper chelators for the treatment of wilson's disease.
The present disclosure is directed to overcoming these and other deficiencies in the art.
Disclosure of Invention
In one aspect, a method of forming a copper-containing complex is disclosed, comprising contacting a copper-containing sample with a compound of formula I:
wherein R is-OH or-O-CH3. In some embodiments, the copper-containing sample is a non-biological solution or suspension. In other embodiments, the copper-containing sample is a biological solution, suspension, tissue, or organism. For example, contacting the sample can comprise administering the compound to the subject. In another example, administration of the compound reduces the cytotoxic effect of copper. In yet another example, the subject is a human diagnosed with a disorder associated with an elevated physiological level of copper. In one example, the disorder is wilson's disease. Another embodiment comprises inhibiting the catalytic activity of an enzyme by forming a copper-containing complex, and the enzyme is selected from the group consisting of pyruvate kinase m (pkm), mitochondrial adenylate kinase 2(AK2), creatine kinase B (ckb), p21 activated kinase (PAK), TP53 regulated kinase (TP53RK), phosphoglycerate kinase 1(PGK1), pyridoxal kinase (PDXK), U-type mitochondrial creatine kinase (CKMT1B), mitogen activated protein kinase (MEK), tyrosine kinase CSK, protein tyrosine phosphatase 1B (PTP1B), and any combination of two or more of the foregoing.
In another aspect, a method of inhibiting the enzymatic activity of a kinase in a sample is disclosed, comprising contacting the sample with a compound of formula I:
wherein R is-OH or-O-CH3And wherein the kinase is selected from the group consisting of pyruvate kinase m (pkm), mitochondrial adenylate kinase 2(AK2), creatine kinase b (ckb), p21 activated kinase (PAK), TP53 regulated kinase (TP53RK), phosphoglycerate kinase 1(PGK1), pyridoxal kinase (PDXK), U-type mitochondrial creatine kinase (CKMT1B), mitogen activated protein kinase (MEK), tyrosine kinase CSK, and any combination of two or more of the foregoing. In one embodiment, the sample comprises a subject in need of drug treatment, and contacting the sample comprises administering the compound to the subject. In one example, the subject has gastric cancer. In another example, the subject has HER2 negative breast cancer. For example, a HER2 negative cancer may be an estrogen receptor negative cancer, a progestin receptor negative cancer, or a triple negative breast cancer. In another example, the subject has triple negative breast cancer. Other examples include administering AZD6244 to a subject. In one example, administration of the compound and AZD6244 to a subject has a therapeutic effect, and the therapeutic effect is greater than the therapeutic effect of administration of the compound to a subject without administration of AZD6244 and the therapeutic effect of administration of AZD6244 to a subject without administration of the compound. For example, the therapeutic effect can include inhibiting tumor growth, inhibiting metastasis of cancer cells, stimulating death of cancer cells, or any combination of two or more of the foregoing. In yet a further example, the compound is administered to a subject in need of drug treatment who previously administered AZD 6244. For example, contacting the sample with the compound can include inhibiting tumor growth, inhibiting metastasis of a cancer cell, stimulating death of a cancer cell, or any combination of two or more of the foregoing.
In yet another aspect, methods of administering to a subject a pharmaceutical composition comprising copper complexed with a compound of formula I:
wherein R is-OH or-O-CH3. In an implementation methodWherein administering the pharmaceutical composition comprises decreasing the body mass index of the subject. In another embodiment, administering the pharmaceutical composition comprises enhancing a physiological response to one or more hormones, and the one or more hormones is insulin, leptin, or both. In yet another embodiment, the subject has cancer, and the cancer is gastric cancer or HER2 negative breast cancer, and administering the pharmaceutical composition comprises inhibiting tumor growth, inhibiting metastasis of cancer cells, stimulating death of cancer cells, or any combination of two or more of the foregoing. In some examples, the cancer may be HER2 negative breast cancer, wherein the HER2 negative breast cancer is estrogen receptor negative, progestin receptor negative, or triple negative breast cancer. Some embodiments further comprise administering AZD6244 to the subject. In some examples, wherein the administration of the compound and AZD6244 to the subject has a therapeutic effect, and the therapeutic effect is greater than the therapeutic effect of the administration of the compound to the subject without the administration of AZD6244 and the therapeutic effect of the administration of AZD6244 to the subject without the administration of the compound. For example, the therapeutic effect can include inhibiting tumor growth, inhibiting metastasis of cancer cells, stimulating death of cancer cells, or any combination of two or more of the foregoing. In yet a further example, the pharmaceutical composition is administered to a subject in need of drug treatment who previously administered AZD 6244. In still other examples, administering the pharmaceutical composition comprises inhibiting tumor growth, inhibiting metastasis of a cancer cell, stimulating death of a cancer cell, or any combination of two or more of the foregoing. In another embodiment, administering the compound comprises inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B) in the subject.
In another aspect, a pharmaceutical composition comprising copper complexed with a compound of formula I is disclosed:
wherein R is-OH or-O-CH3. In one embodiment, the composition comprises a pharmaceutically acceptable excipient.
Drawings
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:
FIG. 1 shows the use of DiFMUP as substrate, increasing concentrations of DPM-1001 (squares) and MSI-1436 (circles) versus the phosphatase PTP1B in elongated form1-405Inhibiting effect.
FIG. 2 shows the effect of increasing concentrations of DPM-1001 (squares) and MSI-1436 (triangles) tested against the PTP1B double mutant S372P/L192A PTP 1B.
FIG. 3 shows PTP1B incubated with DPM-1001 (1. mu.M) for 15min1-405(circles) or PTP1B1-321(squares) (100nM) PTP activity. The complex was diluted 100-fold and activity was monitored for 240 minutes. The results were compared to those for PTP1B1-405(circles) or PTP1B1-321(squares) the measurements made with MSI-1436 were compared and are shown in grey.
FIG. 4 shows the time dependence of DPM-1001 on the inhibition of PTP1B in both the long (black bars) and short (gray bars) forms.
FIG. 5 shows PTP1B in the absence (circle) and presence (triangle) of DPM-10011-394The elution profile of (3).
FIG. 6 shows the presence of H or no H (hollow bar)2O2DPM-1001 vs PTP1B in the case of the degrading enzymes catalase (grey bars) and peroxiredoxin reductase (black bars)1-405Time dependence of inhibition of (a).
FIG. 7 shows DPM-1001(1mM) reacted with CuSO4(8mM) and the reaction mixture analyzed by ESI-MS. The inset shows the isotopic pattern analysis of the peak at 727.5 m/z.
FIGS. 8A and 8B show the complex [ Cu (formula I) SO4]The proposed structure of (1).
FIG. 9 shows increasing concentrations of radiolabeled copper for DPM-1001 titration (II)64Cu)。
FIG. 10 shows the incremental concentration of Cu-DPM-1001 complexes versus WT PTP1B (circle), H320A/H331APTP1B1-405(Square) and H320A/H331A/S372P PTP1B1-405(triangle) effect.
FIG. 11 shows a diagram for PTP1B1-405、PTP1B1-321And increased concentration of the H320A/H331A mutant PTP1B titration64Cu-DPM-1001 complex.
FIG. 12 shows a diagram for a set of PTPs (PTP1B)1-405(circle), PTP1B1-321(squares), TCPTP (diamonds), SHP2 (open circles), LAR (open squares), PTP α (upward triangles), PTP σ (downward triangles), and PTEN (open diamonds)) titrated for increased concentration64Cu-DPM-1001 complex.
FIG. 13 shows the effect of DPM-1001 on body weight. From 10 weeks of age until study termination (18 weeks of age), obese male mice fed High Fat Diet (HFD) (C57bl6/J) were treated daily with DPM-1001(5mg/kg, or oral or intraperitoneal) and body weights were compared to saline treated obese mice (n ═ 10 per group).
Figure 14 shows the effect of treatment with saline or DPM-1001 on HFD-fed 14-week-old male mice administered D-glucose (2mg/g body weight) and monitored blood glucose (n ═ 10 per group). Statistical analysis was performed using two-way analysis of variance (2-way ANOVA) (. p <0.05,. p < 0.01).
Figure 15 shows the blood glucose effect of treatment with saline or DPM-1001 on HFD-fed 14-week-old male mice (per group, n-10) dosed with insulin (0.75mU/g body weight). Statistical analysis was performed using two-way analysis of variance (2-way ANOVA) (. p < 0.05).
Figure 16 shows insulin-induced tyrosine phosphorylation of IR- β in 14-week-old mice treated orally or Intraperitoneally (IP) with saline or DPM-1001. For insulin stimulation, animals were treated with insulin (0.75mU/g, IP) for 15 minutes. Mean ± s.e.m. statistical analysis was performed using two-way analysis of variance (2-way ANOVA) (xp < 0.01).
Figure 17 shows leptin-induced tyrosine phosphorylation of JAK2 in hypothalamic tissue lysates of 14-week-old mice treated with saline or DPM-1001 orally or Intraperitoneally (IP). For leptin stimulation, animals were treated with leptin (1mg/kg, sc) for 15 minutes. Mean ± s.e.m. statistical analysis was performed using two-way analysis of variance (2-way ANOVA) (xp < 0.01).
FIG. 18 shows the ESI-MS spectra for complex formation between DPM-1001(1mM) and the indicated metal (8 mM).
FIG. 19 shows the survival of control cells with (upward triangles) or without (circles) DPM-1001, and ATP7B knockdown cells (ATP7B-KD1) with (gray, downward triangles) and without DPM-1001 (squares), measured at copper concentrations increasing from 0-1.5 mM.
Figure 20 shows the survival of control fibroblasts with (upward triangles) and without (downward triangles) DPM-1001, compared to the survival of fibroblasts derived from wilson's disease patients (GM00032 (upper panel)) with (grey, round) and without (square) DPM-1001, measured at copper concentrations increasing from 0-1.5 mM.
Figure 21 shows the survival of control fibroblasts with (upward triangles) and without (downward triangles) DPM-1001, compared to the survival of fibroblasts derived from wilson's disease patients (GM00033 (middle and small panels)) with (grey, round) and without (square) DPM-1001, measured at copper concentrations increasing from 0-1.5 mM.
Figure 22 shows the survival of control fibroblasts with (upward triangles) and without (downward triangles) DPM-1001, compared to the survival of fibroblasts derived from wilson's disease patients with (grey, round) and without (square) DPM-1001 (GM05257 (lower panel)), measured at copper concentrations increasing from 0-1.5 mM.
Figure 23 shows rhodanine (rhodanine) staining levels of liver tissue from stained TX mouse models of untreated or DPM-1001 treated animals.
Fig. 24 shows copper levels in (a) liver, (B) brain and (C) kidney of wild type (black bars) or TX (grey bars) mice measured using ICP-MS after treatment with saline, DPM-1001(5mg/kg, oral once every three days) or tetrathiomolybdate (TTM) (5mg/kg, intraperitoneal daily) for two weeks. Panel (D) represents the copper levels in the faeces of these animals.
Figure 25 shows levels of metal thionein in the liver (left) or brain (right) of wild type (black bars) and TX (gray bars) mice treated with saline, DPM-1001 or tetrathiomolybdate (TTM).
Figure 26 shows the survival of control fibroblasts with (upward triangles) and without (downward triangles) DPM-1001, compared to GM12158 survival of fibroblasts from wilson's disease patients with (grey, round) and without (square) DPM-1001, measured at copper concentrations increasing from 0-1.5 mM.
Figure 27 shows the survival of control fibroblasts with (upward triangles) and without (downward triangles) DPM-1001, compared to the survival of GM05798(B) fibroblasts from wilson patients with (grey, round) and without (square) DPM-1001, measured at copper concentrations increasing from 0-1.5 mM.
Figure 28 shows the survival of control fibroblasts with (upward triangles) and without (downward triangles) DPM-1001 measured at copper concentrations increasing from 0-1.5mM compared to the survival of GM11778(C) fibroblasts from wilson patients with (grey, round) and without (square) DPM-1001.
FIG. 29 shows survival within 12 months for Wild Type (WT) and TX Wilson's Disease (WD) mice treated with saline or DPM-1001(2 mg/kg).
FIG. 30 shows copper levels in the liver of WT (black bars) and TX (gray bars) mice treated with saline, DPM-1001(2mg/kg) or DPM-1003(2mg/kg) once every three days using ICP-MS.
FIG. 31 shows the effect of DPM-1001 and DPM-1003 on SUM159 cell viability.
FIG. 32 shows the effect of DPM-1001 and DPM-1003 on CAL120 cell viability.
FIG. 33 shows the effect of DPM-1001 and DPM-1003 on HCC38 cell survival.
FIG. 34 shows the effect of DPM-1001 and DPM-1003 on HS578T cell survival.
FIG. 35 shows the effect of DPM-1001 and DPM-1003 on MCF7 cell viability.
FIG. 36 shows the effect of DPM-1001 and DPM-1003 on MDA-MB-231 cell viability.
FIG. 37 shows the effect of DPM-1001 and DPM-1003 on MDA-MB-361 cell viability.
FIG. 38 shows the effect of DPM-1001 and DPM-1003 on MDA-MB-468 cell viability.
FIG. 39 shows the effect of DPM-1001 and DPM-1003 on 4T-1 cell survival.
FIG. 40 shows the effect of DPM-1001 and DPM-1003 on BT-474 cell viability.
FIG. 41 shows the effect of DPM-1001 and DPM-1003 on the survival of SKBR3 cells.
FIG. 42 shows the effect of DPM-1001 and DPM-1003 on MCF10A cell viability.
Figure 43 shows the removal of copper by DPM-1001 in TNBC cells.
FIG. 44 shows the inhibition of tumor size by DPM-1001, but not DPM-1003.
FIG. 45 shows the inhibition of tumor size by DPM-1001, but not DPM-1003.
FIG. 46 shows the inhibition of MDA-MB-231 cell viability by DPM-1001.
Figure 47 shows the survival of HCC70 cells inhibited by treatment with DPM-1001 in combination with AZD 6244.
FIG. 48 shows the inhibition of survival of MDA-MB-468 cells by treatment with DPM-1001 in combination with AZD 6244.
FIG. 49 shows the survival of SUM149 cells inhibited by treatment with DPM-1001 in combination with AZD 6244.
FIG. 50 shows the inhibitory effect of DPM-1001 on SNU116 cell viability.
FIG. 51 shows the inhibitory effect of DPM-1001 on KATOIII cell survival.
FIG. 52 shows the inhibitory effect of DPM-1001 on SNU5 cell viability.
FIG. 53 shows the inhibitory effect of DPM-1001 on AGS cell survival.
FIG. 54 shows the inhibitory effect of DPM-1001 on MDA-MB-468 cell survival.
Fig. 55 shows the inhibitory effect of DPM-1001 on survival of N87.
FIGS. 56A and 56B show interaction with CuSO4(80mM) and CuCl, respectively2(8mM) reacted DPM-1003(1mM), and the reaction mixture was analyzed by ESI-MS.
Detailed Description
Disclosed herein are compounds that can form complexes with copper, or such compounds complexed with copper and uses thereof. Such compounds may be in the form of pharmaceutical compositions and may be administered to a patient in need of drug treatment. In some examples, the compound is administered to a sample, such as a human or animal subject, a sample of biological tissue or cells, or a non-biological sample, to form a complex of the compound with copper in solution. For example, the compounds can be used to chelate copper to prevent or reduce the binding of copper to other molecules in the sample. In other examples, the compound may be administered with or without first being formulated as a complex with copper, such that the complex of the compound with copper that is administered or formed upon administration of the compound to a copper-containing sample may have a therapeutic effect, such as by binding and inhibiting an enzyme. In some examples, the inhibitory effect of a compound on enzymatic activity may be more durable than the effect a compound may have on enzymatic activity when not complexed with copper.
As disclosed herein are compounds of formula I:
wherein R is-OH or-O-CH3. Such compounds are capable of forming complexes with copper. As an example of a compound which inhibits the enzyme protein tyrosine phosphatase 1B (PTP1B), wherein R is-O-CH3Is disclosed in U.S. patent No. 9,546,194. However, a surprising finding as disclosed herein and not previously practiced or known is that the compounds of formula I form complexes with copper with an affinity much higher than any binding affinity for other metal ions, whereas the compounds of formula I do not form complexes with such other metal ions. Furthermore, when complexed with copper, the compound-copper complex of formula I can bind to and thereby inhibit the phosphatase activity of PTP 1B. Inhibition of enzymatic Activity of PTP1B by a Compound-copper Complex disclosed herein, such as the Compound-copper Complex of formula IThe duration of action may be much longer than the duration of inhibition achieved by binding of a structurally similar PTP1B inhibitor to PTP1B that is achieved by binding of the compound to PTP1B or that does not form a complex with copper when the compound is not complexed with copper.
When administered to an animal or human subject, the chemical structure of the compounds of formula I can be altered by metabolic processes to form metabolites. For example, -O-CH at the R position may be converted by metabolic processes in the body3Changing to-OH at the R position. As also disclosed herein, the compounds of formula I can form complexes with copper and can be used as or in the formation of copper-compound complexes, whether R is-O-CH3or-OH. The skilled artisan will appreciate that under physiological conditions, the hydroxyl hydrogen atom at the R-position may be dissociated from the compound, or the hydrogen atom may associate with a nitrogen elsewhere in the compound, such as in the azaalkyl chain attached to the pyrimidine ring, to form an internal acid, and all such compounds will be included in formula I of the present disclosure.
In certain examples, formula I may have the following structure:
wherein R is-OH or-O-CH3. When R is-O-CH3This compound is referred to herein as DPM-1001. When R is-OH, the compound is referred to herein as DPM-1011.
In other examples, formula I may have the following structure:
wherein R is-OH or-O-CH3. When R is-O-CH3This compound is referred to herein as DPM-1013. When R is-OH, this compound is referred to herein as DPM-1015.
In other examples, formula I may have the following structure:
wherein R is-OH or-O-CH3. When R is-O-CH3When such a compound is referred to herein as DPM-1014. When R is-OH, the compound is referred to herein as DPM-1016.
In other examples, formula I may have the following structure:
wherein R is-OH or-O-CH3. In other examples, combinations of two or more of the foregoing formula I and/or other stereoisomers of formula I may be present in different relative ratio combinations with each other, some or all having R as-OH and some or all having R as-O-CH3。
As also disclosed herein, other chemical structures closely related to the compounds of formula I do not form complexes with copper. For example, trodus sequemine, also known as MSI-1436, or 3-N-1 (spermine) -7, 24-dihydroxy-5-cholestane 24-sulfate, is structurally related to DPM-1001. Although DPM-1001 closely resembles the structure of MSI-1436, MSI-1436 does not form a complex with copper. Furthermore, MSI-1436 was able to inhibit the enzymatic activity of PTP1B, similar to DPM-1001 as disclosed herein. However, as also disclosed herein, the duration of inhibition of the enzymatic activity of PTP1B by a compound of formula I complexed with copper, such as DPM-1001, is of significantly longer duration than the inhibitory effect of MSI-1436 on the enzymatic activity of PTP 1B. MSI-1436 has been shown to be a reversible inhibitor of PTP 1B. For example, PTP1B activity recovered in 60 minutes or less when samples containing PTP1B and MSI-1436 were diluted 100-fold, whereas PTP1B activity was still inhibited after similar treatment for at least 240 minutes for samples containing PTP1B and complexes of DPM-1001 with copper. Furthermore, the compounds of formula I have surprisingly higher potency in inhibiting the enzymatic activity of PTP1B, which is enhanced by pre-incubation of the compound with PTP 1B. Potency (100nm IC) of a compound of formula I (such as DPM-1001) when preincubated with PTP1B50) Efficacy (600nm IC) compared to MSI-1436 preincubated with PTP1B50) Much higher. Thus, surprisingly, compounds of formula I complexed with copper (such as DPM-1001) are substantially more potent inhibitors of PTP1B, with significantly longer effective duration, than other compounds having similar chemical structures.
As also disclosed herein, complexes of compounds of formula I, such as DPM-1001, with copper have surprisingly been found to have antitumor activity. MSI-1436 has previously been shown to be toxic to certain types of breast tumor cells. Such effects of MSI-1436 may be associated with an inhibitory effect of MSI-1436 on PTP1B activity in certain types of breast cancer cells. For example, some types of breast cancer cells express the protein HER2 (also known as the receptor tyrosine-protein kinases erbB-2, CD340, proto-oncogene Neu, erbB2), while expression of HER2 stimulates cancer phenotype and tumor growth. PTP1B is overexpressed in certain HER2 positive breast cancer cells, and a lack of expression of PTP1B has been shown to prevent breast tumor development caused by HER2 overexpression. Thus, MSI-1436 is being investigated for use as HER2+Use in the treatment of cancer in a breast cancer patient.
However, in contrast to HER2+ breast cancer, the general role of PTP1B in cancer itself is not clear. For certain types of cells, unlike HER2+ breast cancer cells, PTP1B inhibits promotion and not the cancerous phenotype. Thus, the ability of a compound to inhibit tumor growth or tumor cell survival cannot be predicted based solely on whether it inhibits PTP1B activity. Also, considering the relationship between HER2 expression and PTP1B activity in promoting tumor growth, for breast cancer cells that do not express HER2, it would not be predicted that PTP1B inhibitors would inhibit tumor growth or tumor cell survival. For example, the compound referred to herein as DPM-1003 is the same as DPM-1001, except that the nitrogen atom of the pyrimidine ring of DPM-1003 is at the 3-position of the pyrimidine ring, rather than the 2-position of the pyrimidine ring. DPM-1003 can inhibit PTP1B (K)i2 μ M). However, as disclosed herein, DPM-1003 does not inhibit the survival of triple negative breast cancer cells, which are breast cancer cells that do not express HER2 and do not express either an estrogen receptor or a progestin receptor. The lack of inhibition of survival of triple negative breast cancer cells by DPM-1003 supports the traditional understanding that PTP1B is inhibited to decreaseLow survival of breast cancer cells or preventing growth of breast cancer or reducing the size of tumors, breast cancer cells must express HER 2.
However, DPM-1003 and compounds of formula I such as DPM-1001 differ from each other in that DPM-1003 does not bind or form complexes with copper with high affinity. As also disclosed herein, triple negative breast cancer cells have elevated levels of intracellular copper, and elevated levels of copper transporters responsible for the import of copper into the cell. As also disclosed herein, contacting triple negative breast cancer cells with a compound of formula I (such as DPM-1001) reduces copper levels in the cells, possibly due to the ability of the compound to bind and form complexes or chelate copper with high affinity to copper, while contacting such cells with DPM-1003 does not reduce copper levels, possibly due to the inability of DPM-1003 to bind or form complexes with high affinity to copper. Surprisingly, as disclosed herein, formation of a compound-copper complex by contacting triple negative breast cancer cells with a compound of formula I, such as DPM-1001, effectively inhibits survival of triple negative breast cancer cells. In addition, formation of complexes of the compound of formula I with copper by administration of DPM-1001 to xenograft triple negative breast cancer tumor mice reduced the size of such tumors, while administration of DPM-1003 to such animals did not reduce tumor size or inhibit tumor growth. While it is not surprising that DPM-1003 does not have an inhibitory effect on the survival of HER2 negative breast cancer cells (such as triple negative breast cancer cells) nor on the growth of tumors of such cells, given the conventional understanding that an anti-cancer effect that inhibits PTP1B activity in breast cancer would require the expression of HER2 in cancer cells, it is disclosed herein that the inhibitory effect of forming a compound-copper complex by contacting triple negative breast cancer cells with a compound of formula I, such as DPM-1001, is unexpected and has not previously been shown or performed. As also disclosed herein, formation of complexes of compounds of formula I, such as DPM-1001, with copper by administration of such compounds to gastric cancer cells also inhibits survival of such cells. These effects indicate that DPM-1001 can be administered to treat cancer.
Chelation refers to the binding of a compound to a metal ion such that the metal ion binds with high affinity to the compound and tends to form a complex with the compound and remain bound thereto, rather than being present in solution as a free metal ion with the compound. The compounds of formula I chelate copper by binding with high affinity to copper and forming a complex therewith, so that copper tends not to be released from the complex. As disclosed herein, such complexes are formed under physiological conditions, such as when administered to cells in culture or mammals such as rodents, and will form when administered to a human subject. As also disclosed herein, such complexes of the compounds of formula I with copper also form in non-biological solutions. A non-biological solution or sample is a sample that is not taken from a living body or a previous living body and to which no living cells or tissues or body fluids have been purposely added. A non-biological sample may be a solution from which copper needs to be removed or to which copper levels need to be measured, but which is not extracted from a living organism or a previously living organism and to which no living tissue or cells or body fluids have been purposely added. While the sample may include trace contaminating amounts of biological tissue, such as unexpected trace amounts of cells or bodily fluids, the skilled artisan will recognize that such a sample will be a non-biological sample or non-biological solution if not extracted or obtained from a living subject, or to which tissue or cells or bodily fluids taken from a living subject are not added, or to which or in which cultured cells are added or grown.
As also disclosed herein, the formation of complexes of compounds of formula I, such as DPM-1001, with copper can inhibit the enzymatic activity of various kinases. Because DPM-1001 can form complexes with copper and because it can reduce the copper content in cells, presumably by forming complexes with cellular copper, administration of a compound of formula I to cells can sequester copper in such cells and prevent or reduce copper binding to other cellular molecules. If copper binding is important for the function of such molecules, then contacting such cells with such copper binding compounds and forming their complex with copper would be considered to inhibit the activity of such molecules. As also disclosed herein, many kinases bind copper. For example, as disclosed herein, pyruvate kinase m (pkm), mitochondrial adenylate kinase 2(AK2), creatine kinase b (ckb), p21 activated kinase (PAK), TP53 regulated kinase (TP53RK), phosphoglycerate kinase 1(PGK1), pyridoxal kinase (PDXK), U-type mitochondrial creatine kinase (CKMT1B), mitogen activated protein kinase (MEK), and tyrosine kinase CSK all bind copper.
As also disclosed herein, binding of copper promotes kinase activity of PAK. For example, exposure of triple negative breast cancer cells to high levels of copper results in increased levels of phosphorylated PAK, and phosphorylated targets and signaling molecules downstream of PAK in the PAK activation signaling cascade, such as increased phosphorylated c-Raf and increased phosphorylated Bad, suggesting increased PAK kinase activity via increased binding of PAK to copper. Phosphorylation of Bad promotes cell survival by preventing Bad from affecting pro-apoptotic functions. Thus, the increased binding of copper to PAK, which results in increased PAK activity, promotes cell survival by decreasing the phosphorylation and decreasing the inactivation of Bad. Given the elevated levels of copper in triple negative breast cancer cells, copper can promote survival of triple negative breast cancer cells by stimulating the activity of the PAK signaling cascade and thereby inhibiting apoptosis and cell death of such cancer cells. As also disclosed herein, by, for example, administering DPM-1001 to triple negative breast cancer cells, a complex of the compound of formula I with copper is formed, thereby sequestering copper and preventing copper from binding to PAK and preventing increased PAK activity, decreasing the activity of PAK, and thus inhibiting the survival of triple negative breast cancer cells.
As also disclosed herein, copper binds MEK. Abnormal activity of MEK and abnormal activity of the Ras mitogen-activated protein kinase (MAPK) signaling cascade stimulated by MEK are associated with a variety of cancers. Elevated MEK and Ras-MAPK activities can stimulate cancer cell survival and promote cell proliferation and cell migration functions associated with metastasis. Thus, inhibition of the MEK and Ras-MAPK cascade may prevent certain forms of cancer. For example, in some, but not all, forms of triple negative breast cancer, treatment with the MEK inhibitor AZD6244 (also known as semetinib (selumetinib) or 6- (4-bromo-2-chloroanilino) -7-fluoro-N- (2-hydroxyethoxy) -3-methylbenzimidazole-5-carboxamide) inhibits cell survival. As disclosed herein, cancer cell survival was significantly inhibited in triple negative breast cancer cells treated with AZD6244 at concentrations at which such treatment did not inhibit cancer cell survival by administering DPM-1001 at concentrations at which it itself did not inhibit cancer cell survival to form complexes of the compound of formula I and copper in such cells. In other words, as disclosed herein, co-administration of AZD6244 at a dose that does not inhibit cell survival when administered on its own with DPM-1001 at a dose that does not inhibit cell survival on its own inhibited survival of triple-negative breast cancer cells. Administration of a compound of formula I to such cells will form a compound-copper complex, such as by chelating cellular copper, thereby reducing the amount of copper available to bind MEK. Thus, as disclosed herein, the formation of complexes of the compounds of formula I with copper by administration of such compounds as DPM-1001 may be protective against cancer by inhibiting the activity of MEK and the Ras-MAPK signaling pathway, and thus may be a treatment for such cancers. Furthermore, the compounds of formula I may be adjunctive to treatment with AZD 6244. For example, triple negative breast cancer cells that are non-responsive to AZD6244, or that may be resistant to treatment with AZD6244 due to prior administration of AZD6244, or that have been identified as non-responsive to AZD6244 due to prior administration of AZD6244 being ineffective, for example, by administering DPM-1001 to patients with such cells to form complexes of compounds of formula I with copper may be useful as treatments for such cancers.
As also disclosed herein, chelating copper with a compound of formula I can have a protective effect against cancers caused by dysregulation of the Ras-MAPK pathway due to mutations in the kinases B-Raf or BRAF. BRAF activates the Ras-MAPK cascade, and the ubiquitous mutation of BRAF in many forms of cancer (such as BRAF V600E) leads to increased MEK phosphorylation and increased Ras-MAPK pathway activity in tumor cells. Such BRAF-mediated increases in activity and tumorigenesis are dependent on copper, as such tumorigenic effects of mutant BRAFs (such as V600E BRAF) are diminished in cells lacking the copper transport protein responsible for entry of copper into the cell, and in cells expressing mutant forms of MEK that are unable to bind copper. Furthermore, the tumorigenic effects of V600E BRAF were inhibited by treating the cells with a copper chelator. Furthermore, treatment of certain cancer cells with BRAF inhibitors such as, for example, vemurafenib (N- [3- [5- (4-chlorophenyl) -1H-pyrrolo [2,3-b ] pyridine-3-carbonyl ] -2, 4-difluorophenyl ] propane-1-sulfonamide) can inhibit tumor growth, but some such cells are resistant to vemurafenib, such that prior treatment with vemurafenib results in mutations that desensitize the cells to the anti-tumor effects of vemurafenib. However, despite mutations that produce an anti-tumor effect that allows escape of BRAF inhibition, treatment of such cells with copper chelators reduces tumor growth. Thus, as disclosed herein, chelation of copper and formation of, for example, a complex of DPM-1001 with cellular copper by treatment with a compound of formula I may be a treatment for cancer types represented by interruption of BRAF signaling, such as tumors expressing BRAFV600E or a similar mutation of
As also disclosed herein, angiogenesis can be inhibited by forming a complex of a compound of formula I with copper by administering, for example, DPM-1001. Angiogenesis is important for the survival of many types of tumors, and inhibition of angiogenesis can deprive tumor cells of blood, oxygen, and the nutrient supply required for tumor cell survival, tumor cell growth, or metastasis. Copper is also known to be important for angiogenesis. Thus, administration of copper chelators is known to have a protective effect against some forms of cancer by inhibiting angiogenesis. As disclosed herein, copper can be sequestered by administering a compound of formula I to a subject and forming a complex, e.g., of DPM-1001 with copper, thereby preventing angiogenesis and preventing tumor growth, promoting tumor shrinkage, or preventing metastasis in such a subject.
As also disclosed herein, for example, by administering DPM-1001 to a sample or subject to form a complex of a compound of formula I with copper, may have protection against conditions or disorders that are typical or involve pathologically high levels of copper. For example, wilson's disease involves the accumulation of copper in the body and chronic cytotoxic accumulation of copper in body tissues, as well as an increase in the physiological level of copper relative to its level in a subject without wilson's disease. As the skilled person will recognise, a cytotoxic effect is one that causes cell death or is predicted to cause cell death if sustained for a long time. Symptoms of Wilson's disease include cirrhosis of the liver, hemolytic anemia, neurological abnormalities, and corneal haze. In addition, copper toxicity can occur from exposure to excessive amounts of environmental copper, such as high levels in drinking water or cooking of food products with high acid levels in uncoated copper cookware. Symptoms may include gastrointestinal discomfort such as vomiting, hematemesis or dark stool, hypotension, jaundice, or coma. Chronic pathologically high copper retention can lead to damage of nerve, kidney or liver tissue. Copper chelation is useful in treating such disorders or conditions caused by elevated levels of copper in the body. As disclosed herein, the cytotoxicity or other deleterious effects of such elevated copper may be prevented, treated or reduced, such as by administering, for example, DPM-1001 to an individual or cell or tissue type having or exhibiting elevated levels of copper to form a complex of a compound of formula I and thereby sequester copper in such a subject or sample.
Cells from Wilson's disease patients exhibit greater sensitivity to the cytotoxic effects of increased copper exposure than cells from humans who have never suffered Wilson's disease, and administration of DPM-1001 prevents such cytotoxic effects. Furthermore, in an animal model of wilson's disease (virulent milk mouse model (TX)), TX mice retain and accumulate excessive body and tissue copper levels due to mutations in the copper transporters responsible for copper excretion, TX mice do not live as long as non-TX mice, and TX mice accumulate high levels of copper in various body tissues including the liver and brain. Liver tissue of TX mice also exhibits morphological abnormalities such as hepatocyte enlargement, irregular shape and arrangement, and cytoplasmic lipid droplet enlargement, and elevated expression of metallothionein, a metal binding protein. However, as described herein, treatment of TX mice with DPM-1001(5mg/kg, once every 3 days) can extend their lifespan, inhibit copper accumulation in the liver and brain, prevent elevation of metallothionein expression in the liver, and prevent hepatocyte enlargement in the liver with irregular shape and arrangement, cytoplasmic lipid droplets.
In contrast, treatment of TX mice with DPM-1003 that did not bind copper with high affinity did not affect the level of copper in liver tissue. Another copper chelator, Tetrathiomolybdate (TTM), also prevented copper accumulation in the liver and brain of TX mice (5mg/kg i.p. daily treatment), but resulted in copper accumulation in the kidneys, possibly associated with copper excretion promotion. In contrast, treatment of TX mice with DPM-1001 (oral, 5mg/kg, once every 3 days) did not promote copper accumulation in the kidneys, apparently leading to copper elimination via excretion through the digestive tract, as evidenced by increased copper levels in the feces of TX mice treated with DPM-1001 compared to TX mice treated with TTM or TX mice not treated with DMP-1001. Thus, the compounds of formula I may be administered for the treatment of patients with wilson's disease or other copper toxicity to sequester copper from the body and treat symptoms, diseases or syndromes resulting from excessive exposure to copper, and may be preferred over other copper chelators for this purpose because of the relatively low dosages required and fecal excretion rather than renal accumulation.
In other examples, the formation of complexes of the compounds of formula I with copper by administering the compounds of formula I to a sample or subject may be used to affect the action of various hormones involved in glucose and energy metabolism, such as insulin and leptin, and as a treatment for disorders or conditions associated with the pathological or unbalanced activity of such physiological processes, such as diabetes, hyperglycemia, or obesity. Similarly, administration of a pharmaceutical composition comprising a complex of a compound of formula I with copper to a sample or subject may also be useful for affecting the action of various hormones involved in glucose and energy metabolism, such as insulin and leptin, and as a treatment for disorders or conditions associated with the pathological or unbalanced activity of such hormones, such as diabetes, hyperglycemia, or obesity.
For example, inhibition of PTP1B activity has been identified as a method of treating diabetes, increasing insulin and leptin sensitivity, normalizing blood glucose levels, and treating obesity. As described herein, including above, the compounds of formula I complexed with copper are potent inhibitors of PTP1B activity with a sustained inhibitory effect. As disclosed herein, DPM-1001(5mg/kg, oral or intraperitoneal) administered to high fat diet-fed mice exhibited approximately 5% weight loss (no weight loss in mice not fed a high fat diet) in animal models of obesity and insulin and leptin resistance, showed lower elevation of blood glucose levels in response to glucose injection, and enhanced insulin-lowering effect, indicating enhanced insulin signaling by DPM-1001 treatment. Administration of DPM-1001 (either orally or intraperitoneally) also increased insulin-stimulated phosphorylation of insulin receptors and phosphorylation of AKT in mice, both suggesting increased responsiveness to insulin resulting from DPM-1001 treatment and increased leptin-induced JAK2 phosphorylation in the mouse hypothalamus, suggesting increased responsiveness of leptin receptor signaling to leptin, which is caused by DPM-1001 treatment. Thus, a patient who has been diagnosed with diabetes, hyperglycemia, or obesity may be treated with a compound of formula I complexed with copper, such as by administering DMP-1001 thereto to form a complex with copper present in the subject's body or by administering a pharmaceutical composition comprising a compound of formula I complexed with copper (such as DPM-1001) to promote proper insulin and leptin function and signal transduction, maintain healthy blood glucose levels, and reduce or prevent obesity.
For example, such treatment may reduce the body mass index of the subject, which is calculated as the weight divided by the height squared, and may be in kg/m2Is expressed in units. A compound or pharmaceutical composition comprising a compound of formula I optionally complexed with copper or forming a complex with copper in vivo after administration may be administered to a person having a body mass index between 25 and 30 (typically as characteristic of being overweight) or above 30 (typically as characteristic of being obese) to reduce its body mass index. Such compounds or pharmaceutical compositions may also be administered to persons with a body mass index below 25 to promote physiologically appropriate insulin signaling, leptin signaling, or to reduce body mass index or prevent body mass index gain.
In addition, some diabetic patients show elevated copper levels. Similar to Wilson's patients, elevated copper levels in diabetic patients may have cytotoxic effects and cause tissue or organ damage or dysfunction. The above-described methods for treating patients with elevated copper levels by administering a compound of formula I and thereby forming a complex of such a compound with copper are also suitable for the treatment of diabetic patients. By administering a compound of formula I, such as DPM-1001, to such a patient, a complex may be formed between such a compound and copper, such as by chelating copper within the body, thereby reducing copper levels and preventing cytotoxicity or other adverse effects of pathologically high copper levels present in such a patient.
Also disclosed herein are pharmaceutical compositions comprising a compound of formula I in the form of a copper complex. In some examples, such pharmaceutical compositions may include pharmaceutically acceptable excipients that conform to the formulation of such pharmaceutical compositions, and may be formulated to most effectively administer the pharmaceutical composition to facilitate such formulation.
As disclosed herein, DPM-1001 binds copper with a Kd of 75nM and a stoichiometry of 1 mol/mol. Complexes with copper refer to binding of copper with a Kd of less than 100nM, and include binding of copper with a Kd of 75nM, measured as described in the examples herein. As a non-limiting example, a pharmaceutical composition comprising a compound of formula I complexed with copper comprises, in lyophilized or dried form, such a compound and copper ions such that the dried material is dissolved in a solvent, including that such a compound will bind to copper with a Kd of 100nM or less in solution when administered orally to a subject.
Formulations for administration to a subject include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend on the condition and disorder of the recipient or the intended purpose of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The method may comprise the step of bringing into association a compound of formula I, or a pharmaceutically-acceptable salt thereof ("active ingredient"), with the carrier which constitutes one or more accessory ingredients. In general, the formulation may be prepared by: the active ingredient is combined uniformly and intimately with liquid carriers or finely divided solid carriers or both, and the product is then shaped into the desired formulation, if necessary.
Formulations of the present disclosure suitable for oral administration may be presented as follows: discrete units, such as capsules, cachets, or tablets, each containing a predetermined amount of active ingredient; a powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; or an oil-in-water type liquid emulsion or a water-in-oil type liquid emulsion. The compounds of formula I may also be presented in the form of a bolus, granule or paste. For oral or other administration, a compound of formula I may be suspended in solution or dissolved in a solvent, such as alcohol, DMSO, water, saline, or other solvent, and may also be diluted or dissolved in another solution or solvent, and may, or in some instances may, contain a carrier or other excipient.
In certain embodiments, the compounds of formula I may be combined with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Tablets, troches, pills, capsules and the like may also contain the following: binders such as, for example, gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; a disintegrant such as, for example, corn starch, potato starch, alginic acid, or a combination thereof; lubricants, such as, for example, magnesium stearate; a sweetening agent such as, for example, sucrose, lactose, saccharin or combinations thereof; flavoring agents such as, for example, peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, a carrier, such as a liquid carrier. Gelatin capsules, tablets or pills may be enteric coated. Enteric coatings prevent the composition from denaturing in the stomach or upper intestine where the pH is acidic. Once in the small intestine, the alkaline pH therein dissolves the coating and allows the composition to be released and absorbed by specialized cells, such as epithelial intestinal cells and Peyer's patch M cells. Elixir syrups may contain the active compounds sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds can be incorporated into sustained release formulations and dosage forms.
Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein.
The compounds of formula I may be applied to any sample in which it is desired to bind or chelate copper, or to inhibit the enzymatic activity described herein, or to detect the potential effect or significance of copper utilization. As described above, the sample may comprise a non-biological sample. Or it may be a sample of cells, tissue or body fluid obtained or harvested from a living organism or a previously living organism. A sample may also include a subject, including a human or non-human animal, meaning an organism. For example, a subject may include a human or non-human animal in need of drug treatment. The sample may also include a biological solution, a suspension of biological material or tissue, such as a solution or suspension of components taken from living or previously living cells, or from a culture or tissue culture medium in which living cells or tissue are cultured, or may include a bodily fluid, such as blood, saliva, cerebrospinal fluid, ascites, lymph, plasma, serum, mucus, or other bodily fluids or secretions. The biological sample may be a solution or suspension of organic molecules or other compounds produced by living cells. Samples containing organic molecules synthesized by means other than living cells, tissues or organisms (e.g., by artificial methods), including synthetic copies of other naturally occurring compounds, do not constitute biological solutions or suspensions.
Formulations for parenteral or other administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral or other administration may also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit doses in multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline, Phosphate Buffered Saline (PBS) or the like, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
The term "pharmaceutically acceptable carrier" as used herein refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained by: for example, by using a coating material such as lecithin, by maintaining a desired particle size in the case of a dispersant, and by using a surfactant. These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. Injectable depot forms are prepared by forming a microencapsulated matrix of the drug in biodegradable polymers such as polylactide-polyglycolide, poly (orthoester) and poly (anhydride). Depending on the ratio of the compound of formula I to the polymer and the nature of the particular polymer used, the release rate of the compound of formula I can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers may include sugars such as lactose.
The formula I compound formulation may include different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether sterility is required for the route of administration, such as injection. The invention may be applied by: intravenous, intradermal, transdermal, intrathecal, intraarterial, intraperitoneal, intranasal, intravaginal, intrarectal, topical, intramuscular, subcutaneous, mucosal, oral, topical, regional, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, regional perfusion to directly flush target cells, via a catheter, via lavage, with milk fat, with lipid compositions (e.g., liposomes), or by other methods known to those of ordinary skill in the art or any combination of the above (see, e.g., Remington's pharmaceutical Sciences, 18 th edition, Mack Printing Company, 1990).
The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids and bases, and organic acids and bases. Unless otherwise indicated, reference herein to a compound of formula I, or in particular any such compound, includes reference to a pharmaceutically acceptable salt thereof. When the compounds of the present disclosure are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid (besylate), benzoic acid, betulinic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, ethanedisulfonic acid, ethanesulfonic acid, ethylenediaminetetraacetic acid, formic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalenesulfonic acid, nitric acid, oleic acid, pamoic acid (pamoic acid), pantothenic acid, phosphoric acid, pivalic acid, salicylic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, teoclatic acid, p-toluenesulfonic acid, ursolic acid, and the like. When the compounds contain acidic side chains, pharmaceutically acceptable base addition salts suitable for use in the compounds of the invention include, but are not limited to, metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Additional pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium cations and carboxylate, sulfonate, and phosphonate anions attached to alkyl groups having from 1 to 20 carbon atoms.
The compounds of formula I may be formulated into compositions in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example with the free amino groups of the proteinaceous composition, or with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. After formulation, the solution will be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as formulated for parenteral administration, such as an injection solution, or an aerosol for delivery to the lung, or formulated for digestive administration, such as a drug release capsule or the like.
As used herein, the term "physiologically functional derivative" refers to any pharmaceutically acceptable derivative of a compound of the present invention which, upon administration to a mammal, is capable of providing (directly or indirectly) a compound of the present invention or an active metabolite thereof. Such derivatives, such as esters and amides, will be apparent to those skilled in the art without undue experimentation. Reference may be made to Burger's Medicinal Chemistry And drug discovery, 5 th edition, volume 1: principles and practices (Principles and Practice).
The term "effective amount" as used herein refers to an amount of a compound of formula I agent that can elicit the biological or medical response of a cell, tissue, system, animal or human that is being sought, for example, by a researcher or physician. The term "therapeutically effective amount" refers to an amount that results in an improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount effective to enhance normal physiological function. For use in therapy, a therapeutically effective amount of a compound of formula I, and salts, solvates and physiologically functional derivatives thereof, may be administered as the chemical starting material. In addition, the active ingredient may be present as a pharmaceutical composition.
The pharmaceutical compositions of the present invention comprise an effective amount of a compound of formula I and optionally one or more other agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when properly administered to an animal, such as a human. In light of the present disclosure, one skilled in the art will know the formulation of pharmaceutical compositions comprising a compound of formula I and optionally one or more other active ingredients, as exemplified by remington's pharmaceutical sciences, 18 th edition, macpress, 1990. Further, for animal (e.g., human) administration, it is understood that the formulation should meet sterility, pyrogenicity, general safety and purity standards as required by FDA office of biological standards.
Also according to the invention, compositions of the invention suitable for administration may be provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid (i.e., paste) or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the therapeutic effect of the recipient or the composition contained therein, its use in the administrable compositions for practicing the methods of the invention is suitable. Examples of carriers or diluents include fats, oils, water, salt solutions, lipids, liposomes, resins, binders, fillers, and the like or combinations thereof. The composition may also include various antioxidants to prevent oxidation of one or more components. In addition, the action of microorganisms can be prevented by preservatives such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methyl paraben, propyl paraben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.
According to the present invention, the compounds of formula I may be combined with the carrier in any convenient and practical manner, i.e. by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are conventional to those skilled in the art.
In a particular embodiment of the invention, the composition is intimately combined or admixed with a semi-solid or solid carrier. Mixing may be carried out in any convenient manner, such as milling. Stabilizers may also be added during mixing to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like.
In other embodiments, the invention may relate to the use of a pharmaceutical lipid vehicle composition comprising a compound of formula I and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a wide range of substances that are characteristically insoluble in water and can be extracted with organic solvents. Such a broad range of compounds are well known to those skilled in the art and are not limited to any particular structure as the term "lipid" is used herein. Examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are typically biological substances. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulpholipids, lipids with ether and ester linked fatty acids, and polymerizable lipids and combinations thereof. Of course, compounds other than those specifically described herein that are understood by those of skill in the art to be lipids are also included in the compositions and methods of the present invention.
One of ordinary skill in the art will be familiar with a range of techniques that can be used to disperse the composition in a lipid vehicle. For example, the compound of formula I may be dispersed in a solution containing lipids, dissolved in lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bonded to lipids, contained in lipids as a suspension, contained with or complexed with micelles or liposomes, or associated with lipid or lipid structures by any means known to those of ordinary skill in the art. Dispersion may or may not result in the formation of liposomes.
The actual dosage of the composition of the invention to be administered to a subject (e.g., an animal or human patient) can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, prior or concurrent therapeutic intervention, the patient's idiopathic disease and route of administration, and the purpose of the treatment. Depending on the dose and route of administration, the preferred dose and/or the number of administrations of an effective amount may vary depending on the subject's response or therapeutic purpose. In any case, the physician responsible for administration will determine the concentration and one or more appropriate doses of one or more active ingredients in the composition for an individual subject.
In certain embodiments, the pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise from about 2% to about 75%, or for example, from about 25% to about 60%, and any range derivable therein, by weight of the unit. Naturally, the amount of the compound of formula I in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. One of ordinary skill in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, and other pharmacological considerations, as such, various dosages and treatment regimens may be desirable.
In other non-limiting examples, the dose can further include about 1 microgram/kg body weight, about 5 microgram/kg body weight, about 10 microgram/kg body weight, about 50 microgram/kg body weight, about 100 microgram/kg body weight, about 200 microgram/kg body weight, about 350 microgram/kg body weight, about 500 microgram/kg body weight, about 1 milligram/kg body weight, about 5 milligrams/kg body weight, about 10 milligrams/kg body weight, about 50 milligrams/kg body weight, about 100 milligrams/kg body weight, about 200 milligrams/kg body weight, about 350 milligrams/kg body weight, about 500 milligrams/kg body weight, to about 1000 milligrams/kg body weight, or more per administration, and any ranges derivable therein. In non-limiting examples of ranges derivable from the numbers listed herein, ranges of from about 5mg/kg body weight to about 100mg/kg body weight, from about 5 micrograms/kg body weight to about 500 milligrams/kg body weight, and the like, can be administered in accordance with the numbers above.
The dosage may be modified or selected depending on factors including the purpose of treatment, severity of symptoms, or weight of the individual subject. The daily dose may be administered once daily, or distributed over 2,3, 4, 5, 6, 7, 8 or more administrations per day. The daily dose may be from 10mg to 20g per day. The daily dose may be less than 10mg, for example 5mg or 1mg per day, or in the range between 1 and 5mg or between 5 and 10 mg. The daily dose may be between 10mg and 50mg, or between 50mg and 100mg, or between 100mg and 150mg, or between 150mg and 200mg, or between 200mg and 250mg, or between 250mg and 300mg, or between 300mg and 350mg, or between 350mg and 400mg, or between 400mg and 450mg, or between 450mg and 500 mg. The daily dose may be between 500mg and 600mg, or between 600mg and 700mg, or between 700mg and 800mg, or between 900mg and 1g, or between 1g and 1500mg, or between 1500mg and 2g, or between 2g and 2500mg, or between 2500mg and 3g, or between 3g and 3500mg, or between 3500mg and 4g, or between 4g and 4500mg, or between 4500mg and 5 g. The daily dose may be between 5g and 6g, or between 6g and 7g, or between 7g and 8g, or between 8g and 9g, or between 9g and 10g, or between 10g and 11g, or between 11g and 12g, or between 12 and 13g, or between 13g and 14g, or between 14g and 15g, or between 15g and 16g, or between 16g and 17g, or between 17g and 18g, or between 18g and 19g, or between 19g and 20 g. All subranges within and between any of these ranges are also included in the present disclosure.
In some embodiments, the compounds of formula I may be formulated for administration by the digestive route. The digestive route includes all possible routes of administration where the composition is in direct contact with the digestive tract. In particular, the compounds of formula I may be administered orally, buccally, rectally or sublingually. Thus, the compounds of formula I may be formulated with an inert diluent or with an assimilable edible carrier, or may be enclosed in hard or soft shell gelatin capsules, or may be compressed into tablets, or may be incorporated directly into the diet.
For oral administration, the compound of formula I may alternatively be combined with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual oral preparation. For example, a mouthwash can be prepared that incorporates the compound of formula I in the required amount in a suitable solvent such as a sodium borate Solution (Dobell Solution). Alternatively, the compounds of formula I may be incorporated into oral solutions, such as solutions comprising sodium borate, glycerol and potassium bicarbonate, or dispersed in a dentifrice, or added in therapeutically effective amounts to compositions that may comprise water, binders, abrasives, flavoring agents, foaming agents and humectants. Alternatively, the compounds of formula I may be formulated in the form of tablets or solutions which may be placed sublingually or otherwise dissolved in the oral cavity.
Other formulations suitable for other modes of digestive administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually formulated for insertion into the rectum. After insertion, the suppository will soften, melt or dissolve in the cavity fluid. Generally, for suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, active ingredients in the range of from about 0.5% to about 10%, and preferably from about 1% to about 2%.
In other embodiments, the compound of formula I may be administered by parenteral routes. As used herein, the term "parenteral" includes routes that bypass the digestive tract. In particular, the compounds of formula I may be administered by, for example and without limitation: intravenous, intradermal, intramuscular, intraarterial, intrathecal, subcutaneous, or intraperitoneal.
Solutions of the compounds of formula I as free bases or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under normal conditions of storage and use, these formulations contain preservatives to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form may be sterile and have a degree of fluid ease of injection. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained by: for example, by using a coating such as lecithin, by maintaining a desired particle size in the case of a dispersant, and by using a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
For parenteral administration in aqueous solution, for example, the solution may be suitably buffered if necessary, and the liquid diluent first isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, one skilled in the art will be aware of the sterile aqueous media that may be employed in light of this disclosure. For example, a dose may be dissolved in 1ml of isotonic NaCl solution, or added to 1000ml of subcutaneous perfusion, or injected at the proposed infusion site (see, e.g., Remington's pharmaceutical sciences 15 th edition, pages 1035-. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for the individual subject. In addition, for human administration, the formulations may meet sterility, pyrogenicity, general safety and purity standards as required by FDA office of biological standards.
Sterile injectable solutions can be prepared by incorporating the compound of formula I in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powdered composition may be mixed with a liquid carrier such as, for example, water or saline solution, in the presence or absence of a stabilizing agent.
In other embodiments, the compounds of formula I may be formulated for administration by various other routes, such as topical (i.e., transdermal), mucosal (intranasal, vaginal, etc.), and/or inhalation.
Pharmaceutical compositions for topical administration may include compounds of formula I formulated for pharmaceutical use, such as ointments, pastes, creams or powders. Ointments include all oily, adsorptive, emulsion and water-soluble base compositions for topical application, while creams and lotions are those compositions that contain only an emulsion base. Topically applied drugs may contain penetration enhancers to promote absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerol, alcohols, alkyl methyl sulfoxides, pyrrolidones, and laurocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorbent, emulsion, or water-soluble ointment base. Topical formulations may also contain emulsifying agents, gelling agents, and antimicrobial preservatives as needed to preserve the active ingredients and provide a homogeneous mixture. Transdermal administration of the present invention may also include the use of "patches". For example, a patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
In certain embodiments, the pharmaceutical composition may be delivered by eye drops, intranasal sprays, inhalants, and/or other aerosol delivery vehicles. Methods of delivering compositions directly to the lungs via nasal aerosol sprays have been described. Likewise, drug delivery using intranasal microparticle resins and lysophosphatidylglycerol compounds is well known in the pharmaceutical arts. Also, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix may be employed in accordance with the present disclosure.
The term aerosol refers to a colloidal system of finely divided solids of liquid particles dispersed in a liquefied or pressurized gaseous propellant. The aerosol formulations of the invention for inhalation may consist of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary depending on the pressure requirements of the propellant. Aerosol administration will vary depending on the age, weight and severity and response of the symptoms of the subject.
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