Rotamers of 4-alkyl-5-heteroaryl-3H-1, 2-dithiole-3-thiones
阅读说明：本技术 4-烷基-5-杂芳基-3h-1,2-二硫杂环戊烯-3-硫酮的旋转异构异构体 (Rotamers of 4-alkyl-5-heteroaryl-3H-1, 2-dithiole-3-thiones ) 是由 博米·弗拉姆罗兹 于 2019-09-17 设计创作，主要内容包括：本申请涉及4-烷基-5-杂芳基-3H-1,2-二硫杂环戊烯-3-硫酮的旋转异构异构体。本公开尤其提供4-烷基-5-杂芳基-3H-1,2-二硫杂环戊烯-3-硫酮的旋转异构异构体、其中单个旋转异构形式是通过稳定化获得的此类异构体的复合物和包含过量单个旋转异构体(旋转异构过量)作为稳定化复合物的组合物。本公开还涉及制造和使用以下的方法：此类旋转异构体和复合物,包括用此类旋转异构体或其复合物治疗人或动物患者的方法,例如以预防、治疗或减轻诸如粘膜炎的多种病症的症状和/或出于医学目的提供针对多种器官和组织的氧化损害的保护。(The present application relates to rotamers of 4-alkyl-5-heteroaryl-3H-1, 2-dithiole-3-thiones. The present disclosure provides, inter alia, rotameric isomers of 4-alkyl-5-heteroaryl-3H-1, 2-dithiole-3-thiones, complexes of such isomers in which the single rotameric form is obtained by stabilization, and compositions comprising an excess of the single rotamer (rotameric excess) as a stabilized complex. The present disclosure also relates to methods of making and using: such rotamers and complexes, including methods of treating human or animal patients with such rotamers or complexes thereof, for example, to prevent, treat or alleviate symptoms of various disorders such as mucositis and/or to provide protection against oxidative damage to various organs and tissues for medical purposes.)
1. A composition comprising oltipraz complex, wherein the oltipraz complex has an excess of the (-) rotamer of 4-methyl-5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione (oltipraz).
2. The composition of claim 1, wherein the composition comprises one or more oltipraz complexes having a rotameric excess of the (-) rotamer.
3. The composition of claim 2, wherein at least one of the one or more oltipraz complexes comprises a direct bond between the alpha nitrogen and the electronegative atom of the pyrazinyl ring.
4. The composition of claim 3, wherein the electronegative atom is selected from the group consisting of oxygen, sulfur, and phosphorus.
5. The composition of any one of claims 1-4, wherein the one or more oltipraz complexes comprise hydroxymaleate oltipraz complexes.
6. The composition of any one of claims 1-4, wherein the one or more oltipraz complexes comprise one or more complexes formed by reacting oltipraz with one or more reactants selected from the group consisting of: carboxylic acids or salts thereof, phosphoric acids or salts thereof, alkyl phosphates, aryl phosphates, alkyl sulfonic acids or salts thereof, aryl sulfonic acids or salts thereof, substituted or unsubstituted thiocarboxylic acids or salts thereof, and combinations of one or more of the foregoing.
7. The composition of any one of claims 1-4, wherein the one or more oltipraz complexes comprise one or more complexes formed by reacting oltipraz with one or more reactants selected from the group consisting of: lactic acid, tartronic acid, isothionic acid, isoserine, 2-mercaptoethanesulfonic acid, taurine, propylphosphonic acid, 2-aminoethylphosphonic acid, diethyl dithiophosphate, diethyl thiophosphate, dimethyl phosphate, and combinations thereof.
8. The composition of any one of claims 1-7, wherein the rotameric excess of the (-) rotamer is within a range selected from the group consisting of: 30% -40% excess, 40% -50% excess, 50% -60% excess, 60% -65% excess, 60% -70% excess, 70% -80% excess, 80% -90% excess, and 90% -100% excess.
9. A pharmaceutical composition comprising at least one composition according to any one of claims 1 to 8, wherein said composition is suitable for administration to a human.
10. The pharmaceutical composition of claim 9, wherein the composition is in a form suitable for topical administration, rectal administration, subcutaneous injection, intramuscular injection, intrasternal or intravenous injection, or administration by inhalation.
11. The pharmaceutical composition of claim 9, wherein the composition is in a form suitable for oral administration.
12. The pharmaceutical composition of claim 11, wherein the composition is in the form of a powder, a pill, a tablet, or contained within a capsule.
13. The pharmaceutical composition of claim 11, wherein the composition is in liquid form.
14. Use of a pharmaceutical composition according to any one of claims 9 to 13 for the preparation of a medicament.
15. The use according to claim 14, wherein the pharmaceutical composition is administered orally or buccally.
16. A process for the manufacture of hydroxymaleic anhydride (-) rotamer of oltipraz, said process comprising the steps of:
(i) reacting oltipraz with (+) -diacetyl-L-tartaric anhydride in the presence of an anhydrous polar solvent to form a reaction mixture; and
(ii) acetic acid was added to the reaction mixture.
17. A composition comprising at least a 30% rotameric excess of the (-) rotamer of 4-methyl-5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione (oltipraz).
18. The composition of claim 17, wherein the (-) rotamer of 4-methyl-5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione comprises the hydroxymaleate oltipraz complex.
19. Use of a composition according to claim 17 for the preparation of a medicament.
20. Use of a composition according to claim 18 for the preparation of a medicament.
Technical Field
The present disclosure relates inter alia to rotamers of 4-alkyl-5-heteroaryl-3H-1, 2-dithiole-3-thione (4-alkyl-5-heteroryl-3H-l, 2-dithiole-3-thiones), complexes of such isomers wherein the single rotameric form is obtained by stabilization, and compositions comprising an excess of the single rotamer (rotameric excess) as stabilized complex. The present disclosure also relates to methods of making and using: such rotamers; wherein the single rotamer is a complex obtained by stabilization and present in rotameric excess; compositions comprising rotamers or complexes thereof (including rotameric pharmaceutical compositions); a method for regulating the function of a human oxidative protection gene by using such rotamers or a complex thereof; and methods of treating human or animal patients with such rotamers or complexes thereof (e.g., to prevent, treat, or alleviate symptoms of various disorders such as mucositis, and/or to provide protection against oxidative damage to various organs and tissues for medical purposes).
The disclosure herein relates to novel pharmaceutical formulations, compositions and therapies comprising a population of 4-methyl-5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione (also known as oltipraz) rotamers enriched in a single rotamer that can be stabilized in a complex.
Drawings
FIG. 1 shows oltipraz (4-alkyl-5- (pyrazin-2-yl) -3H-1, 2-dithiole-3-thione).
Fig. 2 depicts oltipraz shown in a configuration in which its two rings are nearly orthogonal to each other.
Figure 3 depicts oltipraz analogs in which the alpha dithiol sulfur atom is substituted with a carbon atom.
Fig. 4A shows the n (+) rotamer of oltipraz.
Figure 4B shows the negative (-) rotamer of oltipraz.
Figure 5 depicts a hydroxymaleic anhydride complex of oltipraz.
The atoms in fig. 2-5 are shown as circles (spheres), with hydrogen labeled with "H", sulfur labeled with "S", nitrogen labeled with "N", or oxygen labeled with "O". The carbon atoms are shown as unlabeled circles (spheres).
Detailed Description
4-methyl-5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione, also known as oltipraz, in crystalline and amorphous forms has been disclosed. Oltipraz has been described as having a variety of properties, for example, as an antimicrobial compound to control schistosomiasis, as an antineoplastic agent, as a chemoprotectant, and as a compound to help control chemical radiation-induced mucositis.
The mechanism of action of chemoprotection and mucositis control effects has been described to be the protection of genes by up-regulation of transcription factor, nuclear factor erythroid 2-related factor 2(Nrf 2). Nrf2 activated by Reactive Oxygen Species (ROS) appears to be a key regulator of oxidative stress gene regulation. Nrf2 is a member of the Cap 'n' Collar family of bZIP proteins. Under normal conditions, Nrf2 binds to its inhibitor, cytoskeleton-associated protein Keap1, which inhibits Nrf2 by promoting its proteasome degradation. After treatment with antioxidants, Nrf2 was released from Keap1 and it initiated upregulation of more than 2000 antioxidant genes.
Oltipraz has been shown to activate a range of oxidative protection genes in an in vitro gene expression assay using pooled human gingival epithelial cells. Gene expression has also been used to manifest itself in subjects with COPD (chronic obstructive pulmonary disorder) as compared to healthy smokers200 differentially expressed oxidative genes are shown, and the significant changes in oxidant response genes observed in vivo were reproduced in vitro using primary bronchial epithelial cells from the same donor. [ Pierrou, S., Broberg, P., O' Donnell, R.A.),K.、Virtala,R.、Lindqvist,E.(2007)Expression of Genes Involved in Oxidative Stress Responses in Airway Epithelial Cells of Smokers with Chronic Obstructive Pulmonary Disease.Am.J.of Respiratory and Critical Care Medicine,175(6),577-587]。
In vitro gene expression has also been studied in Human Corneal Endothelial Cells (HCECs) to determine whether nuclear oxidative DNA damage increases with age. HCECs respond to this damage by up-regulating 4 of their 84 oxidative stress and DNA damage signaling genes in an age-dependent manner. [ Joyce, N.C., Harris, D.L., Zhu, C.C.Age-Related Gene Response of Human nuclear to Oxidative Stress and DNA Damage (2011) nuclear, 52(3), 1641-.
Thus, it is widely accepted that the changes in gene expression observed in vitro cellular assays have been shown to represent physiological oxidative protection conferred to specific organs and tissues.
Chemical structure optimization of oltipraz was used for schistosomiasis control by Quantitative Structure Activity Relationship (QSAR) studies. Although oltipraz has recently shown Nrf2 upregulation activity, it is not known to be a suitable structure or to be in an optimal configuration for Nrf2 upregulation.
As discussed below, it has been found that stable compositions comprising two different rotamers of oltipraz can be prepared. The composition may comprise up to about 80%, 85%, 90%, 95%, 97%, 98%, or 99% rotameric excess of one of the two rotamers, which can be stabilized in the form of an oltipraz complex. Compositions comprising a higher percent of rotational isomerization excess (e.g., greater than 90%, i.e., 95% of one rotamer and 5% of the other rotamer) may be less stable at higher temperatures because the rotating barrier is relatively low. As used herein, "oltipraz complex" means oltipraz that is dissociably associated (covalent or non-covalent) with a molecule or atom. Such compositions comprising a rotameric excess of any one of the individual rotamers may specifically increase upregulation of the transcription factor Nrf2 effect and thus provide oxidative protection in the gastrointestinal system (including in the oral cavity) from chemoradiation and immunotherapy-induced mucositis.
Oltipraz (4-alkyl-5- (pyrazin-2-yl) -3H-1, 2-dithiole-3-thione) consists of two rings, a pyrazinyl ring and a dithiolethione ring, which are linked by a carbon-carbon single bond (fig. 1).
In general, free rotation may occur around such a carbon-carbon (C-C) bond such that the specific orientation of the two rings is not locked and the two rings have been described in X-ray structural publications as being in a twisted planar configuration, with conjugation between unsaturated bonds in each ring allowing for the lowest energy stable state, as shown above in fig. 1. See Wei, C.H., Structure of 4-Methyl-5- (2-pyridyl) -3H-1,2-dithiole-3-thio (Otopraz), A New antiscorscomal drug acta Crystal, (1983), C39,1079-
Using energy minimization computer simulations and applying tinker (r) tools and common parameter sets (e.g., Amber and MMFF), it was found that multi-molecular oltipraz can also take on a stable configuration in which the two rings are nearly orthogonal to each other, as shown, for example, in fig. 2.
Further computer analysis of the analog structure shows that this preferred orthogonal configuration may be due to sulfur hyperconjugation. Substitution of the alpha dithiol sulfur atom with a carbon atom that cannot participate in hyperconjugation results in a planar structure as the minimum energy configuration, with free rotation around the attached C — C single bond, as shown in fig. 3.
Simulations also show that there is an unexpectedly high energy barrier of 14.1kJ/mol between one orientation of two orthogonal rings and its mirror image. This can be attributed to the sulfur hyperconjugation effect that needs to be broken to allow rotation along the bond between the two rings. Thus, the rotamers are a pair of atropisomers, which are due to steric rotation around the bond between the two rings of oltipraz.
Rotamer compositions
Thus, two different rotamers of oltipraz may exist. Each of the two rotamers can be defined as two rings that are orthogonal to each other; the (+) -rotamer is defined by orienting the thione and methyl groups at the top and the alpha nitrogen of the pyrimidine ring to the right, while the (-) -rotamer has the alpha nitrogen of the pyrimidine ring on the left (see fig. 4A and 4B).
It has been found that the preparation of oltipraz complexes in a variety of salt forms as described below results in compositions in which the (+) -rotamer (figure 4A) may be present in a rotameric excess (e.g., up to about 80%). The term "rotameric excess" as used herein means an excess of one rotamer relative to the other rotamer, as generally applied to the term "enantiomeric excess". Thus, "rotameric excess" is described herein as a measure of the purity of the population of oltipraz rotamers as a whole. It reflects the degree to which the sample contains one rotamer in a greater amount than the other rotamer. The meso mixture has a 0% rotoisomeric excess and is completely pure
The pure rotamer has a 100% rotameric excess. Thus, for example, a sample containing 70% of one rotamer and 30% of the other rotamer has a rotameric excess of 40% (70% -30%).
The relative percentage of one isomer relative to the other can be assigned using the relative intensities of the thioketone peaks at about 1200cm-1 and about 440cm-1 in the FTIR analysis. The (+) rotamer showed FTIR thione peaks between 420 and 424 and 1210 to 1215, while the (-) rotamer showed FTIR thione peaks between 432 and 436 and 1200 to 1205.
Examples of oltipraz complexes that can exhibit a rotameric preference for the (+) -rotamer (see example 1 and examples 3-6 below) include salts derived from: groups are added or associated (e.g., reversible addition of HCl, alkyl halides, or acyl halides) to the pyrazinyl ring of oltipraz (e.g., at the alpha nitrogen), resulting in N-substituted pyrazinium ions (the positive charge is most likely concentrated on the alpha nitrogen of the pyrazinyl) and associated anions. Can be caused bySome substituents present on the pyrazine ring that form salts include, but are not limited to, hydrogen, alkyl, alkenyl, alkynyl, acyl, benzoyl or benzoyl, which substituents may be unsubstituted or substituted with other functional groups including, but not limited to, halide (e.g., Cl or F), alkyl (e.g., methyl, ethyl, n-propyl, isopropyl), alkoxy (-OCH), and the like3or-OCH2CH3) Aryloxy groups (e.g., -O-phenyl), and the like. For example, such salts may be formed by reacting oltipraz with HCl. In another example, such salts may be formed by reacting oltipraz with an alkyl, alkenyl, or alkynyl halide (e.g., methyl chloride, methyl iodide, etc.). In another example, such salts can be formed by reaction with an acyl, benzoyl, or benzoyl halide (e.g., a chloride such as acetyl chloride, n-butyl chloride, benzoyl chloride, and the like).
It has also been found that (-) rotamers that can be present in substantial rotameric excess (e.g., up to about 90% or more, including 30% -40% excess, 40% -50% excess, 50% -60% excess, and 60% -65%, 60% -70%, and 70% -80% excess) can be prepared (see example 2 and examples 7-11 below).
The (-) rotamer oltipraz complexes can be prepared, for example, as described in example 2 and examples 7-11, by reacting oltipraz with a variety of carboxylic or phosphoric acids and their salts, alkyl and aryl phosphates, alkyl and aryl sulfonic acids and their salts, substituted and unsubstituted thiocarboxylic acids and their salts. For example, in the case of the hydroxymaleate oltipraz complex, oltipraz is reacted with (+) -diacetyl-L-tartaric anhydride in 5ml of an anhydrous polar solvent (e.g., ultra-anhydrous acetone), followed by the addition of glacial acetic acid. This reaction forms a bond between the α nitrogen of the pyrazinyl ring and the oxygen atom of the maleate salt, essentially simultaneously with the cyclized rearrangement of the diacetyl tartrate to produce the maleic anhydride ring.
Other complexes of oltipraz that may exhibit a rotameric preference for the (-) rotamer include complexes in which a direct bond is formed between the alpha nitrogen of the pyrazinyl ring and an electronegative atom (such as oxygen, sulfur, or phosphorus) sufficient to disrupt the hyperconjugate within the molecule. Such oltipraz complexes can provide up to 100% rotameric excess of (-) rotamer if they can stabilize the (-) rotamer configuration by disrupting the hyperconjugate and/or increasing the stereo strain described above. For example, such oltipraz complexes can provide a rotameric excess of the (-) rotamer in an amount selected from the group consisting of: at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Thus, the rotameric excess of the (-) rotamer may be, for example, an amount selected from the group consisting of: 30% -40% excess, 40% -50% excess, 50% -60% excess, 60% -65% excess, 60% -70% excess, 70% -80% excess, 80% -90% excess, and 90% -100% excess. Such oltipraz complexes include complexes formed by reacting oltipraz with a variety of carboxylic or phosphoric acids and their salts, alkyl and aryl phosphates, alkyl and aryl sulfonic acids and their salts (e.g., substituted and unsubstituted thiocarboxylic acids and their salts, such as lactic acid, tartronic acid, isothionic acid, isoserine, 2-mercaptoethanesulfonic acid, taurine, propylphosphonic acid, 2-aminoethylphosphonic acid, diethyl dithiophosphate, diethyl thiophosphate, dimethyl phosphate). It should be noted that it seems impossible to induce the formation of 5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione into a configuration such as the (-) rotamer of oltipraz by changing the size of the substituent at position 4 of the dithiolethione ring, which is methyl in oltipraz.
Additionally or alternatively, for hyperconjugation of the alpha nitrogen involved in the pyrazinyl ring, hyperconjugation may also be affected by formation of an oltipraz complex with the meta nitrogen of the pyrazinyl ring. Such (-) rotameric complexes may be formed by bonds between a meta nitrogen atom and a carbon, oxygen, sulfur or phosphorus atom. This bond may occur alone or in combination with a complex involving the alpha (ortho) nitrogen atom on the pyrazinyl ring.
Since oltipraz conformers are understood to be distributions in the Boltzmann distribution (Boltzmann distribution) that vary with their relative energies, one way to obtain a higher rotameric excess of the single oltipraz rotamer (or the single rotamer of the oltipraz complex) that is most stable under any given set of conditions (e.g., a particular solvent) involves cooling a solution of the desired oltipraz composition (e.g., below room temperature, e.g., -78 ℃ or less) and providing sufficient time to substantially equilibrate the rotamers. Under such conditions, more molecules will be in a more stable (lower energy) conformation than room temperature. The cooled composition is then dried by evaporation and/or lyophilization, preferably without the use of heat. It has also been found that oltipraz complexes (e.g., salts) in which the (-) -rotamer or (+) -rotamer is in rotameric excess can be substantially purified to a purity of, for example, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, and greater than 99%. The purification process and conditions should be selected to minimize variation in the desired rotameric excess of the composition. For example, the use of temperatures above 40 ℃ or the use of very polar solvents (e.g., DMSO) during purification allows the equilibrium and substantial loss of the rotameric excess to approach or equal 0, while the use of chiral chromatography appears to improve the rotameric excess yield. Thus, exposing the oltipraz complex to a chiral medium under conditions where one rotameric form associates more strongly with the medium than the other rotameric form, allowing time for the complex to equilibrate, and eluting the complex under conditions that are not conducive to rotameric equilibration (e.g., low temperatures), represents a way to enrich the oltipraz complex in the desired configuration.
It has also been found that when complexes (e.g. salts) of the (+) -rotamer and the (-) -rotamer are stored at room temperature (e.g. between 15 ℃ and 30 ℃ and between 40% and 80% relative humidity, such as between 20 ℃ and 25 ℃ and 50% relative humidity), the (+) -rotamer and the (-) -rotamer are oriented not in a transition state, but in a substantially stable configuration. In this context, "substantially stable" means that the concentrations of (+) -rotamer and (-) -rotamer in the composition as measured by FTIR (as discussed below) within 30 minutes after the final purification will not change by more than an amount selected from the group consisting of: 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1%.
Pharmaceutical compositions comprising rotamer compositions
The rotamer compositions described above may be prepared as pharmaceutical compositions as (i) one or more oltipraz complexes (e.g., salts) comprising a rotameric excess of the (+) -rotamer, (ii) one or more oltipraz complexes (e.g., the complexes of example 2 and examples 7-11) comprising a rotameric excess of the (-) -rotamer, or (iii) mixtures of (i) and (ii). The above pharmaceutical compositions (i) - (iii) are referred to herein as "rotamer pharmaceutical compositions". Such rotamer pharmaceutical compositions may be administered as dry or liquid compositions. Formulations of the rotamer pharmaceutical compositions for oral administration may also be presented as mouthwashes, or carbonated liquids, or oral sprays or aerosols, or oral ointments, gels, or creams.
In certain aspects, liquids suitable for formulating rotamer pharmaceutical compositions for oral administration (e.g., buccal administration) can include aqueous and non-aqueous carriers, such as water; brine; a buffer solution; organic solvents, such as alcohols (e.g., ethanol), glycols, aliphatic alcohols; mixtures of water and organic solvents and mixtures of organic solvents (optionally also containing water); lipid-based materials, polysaccharide-based materials, and other vehicles and vehicle components suitable for application to the oral cavity, as well as mixtures of buccal vehicle components as identified above or otherwise known in the art.
Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, microemulsions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents (for emulsions and suspensions), sweetening, flavoring, coloring, perfuming and preservative agents.
Alternative aspects of rotamer pharmaceutical compositions suitable for oral administration of the rotamer pharmaceutical compositions include compositions in the form of: capsules (including sprinkle and gelatin capsules), sachets, sticks, pills, tablets, lozenges, lyophils, powders, granules, implantable compositions or solutions or suspensions in aqueous or non-aqueous liquids, including, for example, compositions suitable for injection or infusion, or oil-in-water or water-in-oil liquid emulsions, or elixirs or syrups, or soft tablets and/or mouthwashes, etc., each containing a predetermined amount of a composition comprising an amount of the rotameric pharmaceutical composition as described herein as the active ingredient. The compositions or compounds may also be administered in a bolus, electuary or paste.
To prepare solid dosage forms for oral administration (capsules (including sprinkle and gelatin capsules), tablets, pills, dragees, powders, granules, and the like), a composition comprising an amount of a rotamer pharmaceutical composition may be mixed with one or more pharmaceutically acceptable carriers and/or any one of the following: fillers or extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, complexing agents, and colorants. Rotamer pharmaceutical compositions may also include components suitable for improving the stability or effectiveness of the formulation. In the case of capsules (including spray capsules and gelatin capsules), tablets and pills, the rotamer pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using known excipients.
Tablets and other solid dosage forms of the rotamer pharmaceutical compositions may optionally be scored or prepared using coatings or shells, such as enteric coatings and other coatings known in the art. They may also be formulated to provide slow or controlled release of the active ingredient therein to provide a desired release profile. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only (or preferentially) in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient may also be in microencapsulated form, if appropriate with one or more of the excipients mentioned above.
Rotamer pharmaceutical compositions for topical administration
In some aspects, the rotamer pharmaceutical compositions may be incorporated into formulations that may be suitable for topical administration, and may include moisturizers, humectants, and other additives known for topical administration. Topical compositions can also be delivered transdermally through a patch applied to the skin, and such patches are well known in the art. One of skill in the art will recognize topical delivery compositions and vehicles that can be used.
Rotamer pharmaceutical compositions for rectal/colonic delivery
In certain aspects, the rotamer pharmaceutical compositions described above can be formulated for rectal administration to provide colon-specific delivery using known methods and compositions. In general, delivery of rotamer pharmaceutical compositions by rectal administration may be achieved by use of suppositories, enemas, ointments, creams or foams. Suppositories are the most commonly used rectal dosage forms and the base is usually fatty in nature, but water-soluble or water-immiscible bases may also be used. To achieve the desired bioavailability, the active ingredient should be in contact with the rectal or colonic mucosa.
Suitable excipients known for use in preparing compositions suitable for rectal administration include, but are not limited to, vehicles, preservatives, surfactants, emulsifiers, mineral oils, propellants, thickeners, lubricants, preservatives, pH adjusting agents, chelating agents, emollients and/or humectants, penetration enhancers, suspension formers or mucoadhesives or combinations thereof. The vehicle may include aqueous, non-aqueous or hydroalcoholic components known to be compatible with rectal and colonic mucosa.
Alternatively, colonic absorption may be accomplished by oral administration of a rotamer pharmaceutical composition designed to release active oltipraz in the colon. Such compositions may be in oral dosage forms, such as pills, capsules or liquids, which provide delayed release until the dosage form is in the colon
Rotamer pharmaceutical compositions and devices for inhalation administration
In other aspects, the rotamer pharmaceutical compositions described above may be delivered via the respiratory tract by providing the composition in inhalable form (e.g., in an inhalation device, in dry powder form or in a liquid carrier). For example, the inhalable composition may comprise the active rotamer pharmaceutical composition provided in a dry powder composition in a dry powder inhaler. See, for example, WO2014177519 and US 20140065219. Alternatively, the inhalable composition may comprise the active ingredient in a liquid carrier, for example ethanol. See, for example, EP2536412 a 2.
Accordingly, the present disclosure also provides a kit comprising (i) a rotamer pharmaceutical composition as described above, and (ii) a device for administering such composition by inhalation. The kit optionally further contains instructions for use.
Device for oral administration
In certain aspects, liquid formulations of rotamer pharmaceutical compositions for oral administration can be prepared and administered using a device that facilitates administration of a single dose of the rotamer pharmaceutical composition. Such devices known in the art may include a cavity or reservoir in which a dry composition and a liquid (e.g., water and/or a non-aqueous solvent) may be mixed and then administered to a patient through an opening in the device. Typically, such devices include a cavity and a chamber separate from the cavity in which the dry powder may reside. Upon administration, the powder is released from the chamber into the cavity or reservoir. In some devices, this is accomplished by breaking a barrier separating the chamber from the cavity or reservoir. The powder is then typically mixed with the liquid in the cavity by shaking, which liquid may be added at an earlier time or at the time. The cavity is of a size sufficient to contain the dry rotamer pharmaceutical composition and an amount of liquid comprising an amount of water and/or a non-aqueous solvent sufficient to allow mixing of the dry rotamer pharmaceutical composition to form a liquid composition. The liquid may be added to the container at the time of packaging to produce a self-contained product comprising the dry composition and the liquid which may be mixed together at the time of application. Alternatively, the container may contain only the dry rotamer pharmaceutical composition and then the liquid is added prior to administration. The liquid may contain flavoring additives as discussed below. Alternatively, other types of packaging that separate dry and liquid components may be used. For example, the powder and liquid may be sealed in 2 form-fill-seal bags, side-by-side or one on top of the other and separated by a rupturable seal. The person administering the drug will then break the seal and mix the contents back and forth between the 2 cells until dissolved or suspended.
Once the composition is substantially homogeneous (e.g., by shaking), it is administered to the patient immediately through an opening in the device created, for example, by untying a portion of the device to expose a cavity containing the liquid mixture. For example, a portion of the device (e.g., the top) may be removed by unscrewing the threaded portion from another threaded portion of the container to expose a cavity containing a liquid mixture that may then be administered to or by the patient. Examples of such devices are provided in U.S. patent 6,148,996, U.S. application 20080202949, and U.S. patent 3,156,369. Such disposable devices may be used for oral administration of the liquid compositions described herein, particularly for the prevention or treatment of oral mucositis or its symptoms as described below.
Accordingly, the present disclosure also provides a kit comprising (i) a rotamer pharmaceutical composition as described above, (ii) a device for oral administration of such composition. The kit optionally further contains instructions for use.
For such devices, the rotamer pharmaceutical compositions may be in dry form. In such cases, the dry composition, which may be present together, for example, in a cell as described above, is mixed with water and/or other liquid solvent (e.g., by exposing the dry composition to a liquid and shaking) prior to application, as discussed above.
Method of treatment
In certain aspects, the rotamer pharmaceutical compositions and devices described above can be used to treat a human or non-human animal patient in need thereof. The patient will typically be a human patient, but the rotamer pharmaceutical compositions of the disclosure may be used to treat a non-human animal, for example for veterinary applications. The compositions of the present disclosure are useful in the prevention or treatment of a wide variety of diseases and conditions, including those known to be treated with oltipraz. Examples of such diseases and conditions include mucositis, HIV, cancer, hepatitis (including HBV and HCV), keratin-based skin diseases including blistering and blistering epidermolysis and related diseases, dermatitis, inflammatory disorders or diseases including endothelial dysfunction and cardiovascular disease, cachexia, weight loss, sepsis, contrast-induced nephropathy, diabetes, obesity, PCOS, steatosis, hyperlipidemia and hypertension, chronic kidney disease, pulmonary fibrosis, hypoxic conditions, chemical-induced lung injury, respiratory distress disorders, anionic interstitial acidosis, nephritis, lupus, interstitial lung disease, graft dysfunction, hepatitis, acute kidney injury, noise-induced stress injury, toxicant intake, retinopathy, neurotoxicity, cancer-induced injury (e.g., ototoxicity), respiratory infection, Autism, conditions involving vasospasm, and conditions that are considered treatable by providing n-acetyl cysteine, injectable reduced glutathione, or known intracellular glutathione enhancers.
Typically, the rotamer pharmaceutical composition is provided to the patient in an effective amount. The term "effective amount" is used herein to refer to an amount of a rotamer pharmaceutical composition sufficient to produce a significant biological response (e.g., a significant reduction in inflammation). Actual dosage levels of the rotamer pharmaceutical compositions can vary to administer amounts effective to achieve the desired therapeutic response for a particular subject and/or application. Of course, the effective amount in any particular case will depend upon a variety of factors including the formulation, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
The term "subject" as used herein includes both human and animal subjects, and thus provides veterinary therapeutic uses in accordance with the present disclosure. The terms "treatment" or "treating" relate to any treatment of an associated condition (e.g., mucositis, inflammatory disorder, or cancer), including, but not limited to, prophylactic and therapeutic treatments. Thus, the terms "treatment" or "treating" include (but are not limited to): preventing development of the associated disorder or condition; inhibiting the progression of the associated disorder; preventing or preventing further development of the associated disorder; lessening the severity of the associated disorder; ameliorating or alleviating symptoms associated with the associated disorder; and causing partial, substantially complete, or complete regression of the associated condition or one or more symptoms associated with the associated condition.
The rotamer pharmaceutical compositions and devices described above are suitable for treating patients who have mucositis or will undergo a treatment that can cause mucositis (e.g., radiation therapy or chemotherapy), for example, in the oral cavity (including in the buccal cavity), in the alimentary canal, in the colon and/or rectum, and/or on the skin. If the mucositis is oral mucositis, the compositions and devices for oral delivery described above may be preferred. For example, such patients may be undergoing or be undergoing chemotherapy and/or radiation therapy, such as radiation treatment of the head and neck region or another region of the body.
The rotamer pharmaceutical compositions and devices described above may be used to accomplish one, more than one, or all of the following beneficial effects in human or non-human animal patients, namely (i) prophylactic prevention or delay of onset of mucositis, including oral mucositis, e.g., mucositis, (ii) treatment of existing mucositis, including oral mucositis, (iii) alleviation of symptoms associated with mucositis, including oral mucositis, (iv) reduction or lessening of severity of existing mucositis, including oral mucositis, (v) promotion of radical or curative treatment of mucositis, including oral mucositis, (vi) reduction of occurrence and/or duration of mucositis, including oral mucositis, e.g., mild, moderate, and severe oral mucositis, (vii) prophylactic prevention or delay of weight loss or onset of cachexia in patients suffering from oral mucositis, (viii) (ix) reducing the amount of weight loss or cachexia experienced by a patient suffering from oral mucositis, and/or (ix) increasing the ability of a patient suffering from oral mucositis to eat through the mouth. Such compositions may also be used to prevent and/or treat patients suffering from dysphagia (difficult to swallow), for example cancer patients, or to delay the onset of or reduce the severity of dysphagia, for example in cancer patients. Such compositions may also be used to prevent and/or treat patients suffering from dry mouth (the subjective feeling of dry mouth), or to delay the onset of dry mouth, reduce the severity of dry mouth, and/or reduce the occurrence of moderate to severe dry mouth. In certain aspects, the disposable devices described above may be used to administer liquid compositions to accomplish one, more than one, or all of the above-mentioned conditions associated with oral mucositis, dysphagia, and dry mouth. Advantageously, the formulation is also non-irritating, well tolerated, palatable (if administered orally), non-cytotoxic, weak or non-sensitizing, non-sensitizing.
Certain aspects herein provide methods of treating mucositis comprising administering to a patient in need thereof a therapeutically effective amount of a rotamer pharmaceutical composition as described herein. The present disclosure also provides a rotamer pharmaceutical composition as described herein for use in treating mucositis. The present disclosure also provides the use of a rotamer pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of mucositis. Administration of the formulation to a patient may be oral, including buccal. The methods of administration described herein may represent a treatment regimen of a predetermined duration (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer). The rotamer pharmaceutical compositions of the present disclosure may be administered once daily, twice daily, three times daily, or as needed (applied) or (administered). Where the patient is undergoing chemotherapy and or radiation therapy, the dose may be administered prior to treatment (e.g., within 1 hour, within 3 hours, within 6 hours, within 12 hours, within 24 hours, or more than 24 hours prior to treatment). For example, administration may begin several days before receiving chemotherapy, radiation therapy, and/or chemoradiation therapy. For example, a patient may begin receiving treatment once daily or more than once daily beginning three days prior to initiation of therapy and then receive daily dosing for a course of treatment (including days not receiving therapy). The administration is given on the day of treatment, e.g., before, e.g., within 1 hour before, e.g., administration of the therapy. Administration may be given at any time of day (e.g., in the morning) on the day of non-treatment. Additionally or alternatively, the dose can be administered post-treatment (e.g., within 1 hour, within 3 hours, within 6 hours, within 12 hours, within 24 hours, or more than 24 hours post-treatment).
Accordingly, the present disclosure provides a method of treating mucositis comprising administering to a patient in need thereof a therapeutically effective amount of a rotamer pharmaceutical composition as described above. The present disclosure also provides a rotamer pharmaceutical composition as described above for use in the treatment of mucositis. The present disclosure also provides the use of a rotamer pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of mucositis. Administration of the rotamer pharmaceutical composition to a patient may be oral administration, including buccal administration.
If a liquid composition is administered, the composition can be administered orally or parenterally (e.g., by subcutaneous, intramuscular, intrasternal, or intravenous injection). If oral administration is employed, the liquid composition may be simply swallowed, or it may be administered by a "swallow and swallow after mouth wash" regimen or a "spit and spit after mouth wash" regimen. By orally administering a composition in liquid form to a patient having oral mucositis or at risk of developing oral mucositis (e.g., undergoing or being subjected to chemotherapy, radiation therapy and/or chemoradiation therapy), the composition can provide therapeutic benefits for mucositis as described above, i.e., it can prophylactically prevent or delay the onset of mucositis, treat existing mucositis, alleviate or reduce symptoms associated with mucositis (e.g., mucositis and bleeding), attenuate or reduce the severity of existing mucositis, shorten the duration of mucositis, and/or promote attenuation, eradication or cure of mucositis. In such cases, liquid compositions comprising negatively charged ingredients (e.g., cationic surfactants or polymers, such as Eudragit RL) can provide another advantage by providing adhesion or association with the oral mucosa, which tends to have a positive charge. The physical and chemical properties of aspects of the compositions described herein may impart features to the formulation such as stability, delivery of the active agent to the mucosa, and ease of administration.
As described above, the rotamer pharmaceutical compositions as described herein may be co-administered with other therapeutic agents, either together or separately, as part of a therapeutic regimen. Such agents include N-acetylcysteine and/or other antioxidants,Pantothenic acid (vitamin B5) or other agents that enhance glutathione synthesis, glutathione (e.g., for topical administration), Medihoney (for topical administration), curcumin (for topical administration) or other NF-. kappa.B inhibitors, melamines (Mesalamine) and/or other anti-inflammatory agents (e.g., for oral or rectal administration of the composition) and superoxide dismutase or prophylactically active O2 -(superoxide) damage of other compounds.
Examples
Certain aspects of the present disclosure are further illustrated by the following examples, which should not be construed as in any way limiting the scope of the appended claims.
Example 1: method for producing hydrochloride salt of octilaprazole (+) rotamer
20mg of synthesized oltipraz powder (ST-617-API, discussed below) was recrystallized using acetonitrile: water and it was added to a 5ml glass vial equipped with a rubber septum. To the vial was added 1ml of anhydrous chloroform at room temperature and the vial was placed on a shaking table until the solution was clear. Using a gas syringe, 5ml of anhydrous hydrochloric acid gas was slowly bubbled into the solution over 5 minutes and the bottle was shaken intermittently. Once all HCl gas was added, the vial was immediately shaken for 5 minutes, the septum removed, and the solvent gently evaporated under a slow steady stream of nitrogen to obtain the hydrochloride complex of oltipraz as an orange/red solid. FTIR: c ═ S peaks 1203/1211 (relative intensity 3:7) and 433/421 (relative intensity 2: 8).
Example 2: process for the manufacture of hydroxymaleic anhydride complexes of the (-) -rotamer of oltipraz
20mg of the synthesized oltipraz powder was recrystallized using acetonitrile-water and added to a 10ml glass bottle equipped with a rubber septum. All moisture was removed from the bottle by nitrogen using a gas syringe. The vial was placed in an ice bath and a pre-cooled (10 ℃) solution of 22mg (+) -diacetyl-L-tartaric anhydride in 5ml of ultra-anhydrous acetone was added thereto, shaken on a shaking table for 5 minutes, and after 5 minutes 0.5ml of glacial acetic acid was added over 30 seconds using a syringe. After intermittent shaking at 0-10 ℃ for 4h, the bottle was warmed to room temperature. The septum was removed and the solvent was gently evaporated under a slow steady stream of nitrogen to obtain hydroxymaleic anhydride complex of oltipraz as an orange solid (see figure 5). The complex was washed 3 times with diethyl ether to remove traces of acetic acid. FTIR C ═ S peaks 1201/1214 (relative intensity 8:2) and 434/423 (relative intensity 8: 2). A 2mg sample of oltipraz complex was ground with KBR and made into standard pellets for FTIR analysis on a Perkin Elmer1600 series FTIR.
Example 3: method for producing acetyl chloride complex of octilaprazole (+) rotamer
20mg of the synthesized oltipraz powder was recrystallized using acetone-water and added to a glass bottle equipped with a rubber septum. To the vial was added 10ml of anhydrous chloroform at room temperature and the vial was placed on a shaking table until the solution was clear. Using a syringe, 5ml of anhydrous acetyl chloride was slowly added to the solution and the bottle was shaken intermittently. Once all the acetyl chloride was added, the vial was immediately shaken for 15 minutes, the septum removed, and the solvent and excess reagents were gently evaporated under a slow steady stream of nitrogen to obtain acetyl chloride complex of oltipraz as an orange solid. FTIR: c ═ S peaks 1202/1212 (relative intensity 3:7) and 432/422 (relative intensity 2: 8).
Example 4: process for producing n-butyl chloride complex of octilaprazole (+) rotamer
20mg of synthesized oltipraz powder was recrystallized using acetone-water and added to a glass vial equipped with a rubber septum. To the vial was added 10ml of anhydrous chloroform at room temperature and the vial was placed on a shaking table until the resulting solution was clear. Using a syringe, 5ml of n-butyl chloride was slowly added to the solution and the vial was shaken intermittently. Once all the n-butyl chloride was added, the vial was immediately warmed to 65 ℃ and shaken for 30 minutes, the septum was removed, and the solvent and excess reagent were gently evaporated under a slow steady stream of nitrogen to obtain n-butyl chloride complex of oltipraz as a red solid. FTIR: c ═ S peaks 1201/1213 (relative intensity 2:8) and 433/421 (relative intensity 2: 8).
Example 5: method for producing methyl iodide complex of octilaprazole (+) rotamer
50mg of the synthesized oltipraz powder was recrystallized using acetone water and added to a pressure rated glass vial equipped with a spiral seal. To the vial was added 20ml of anhydrous chloroform at room temperature and the vial was placed on a shaking table until the resulting solution was clear. Using a syringe, 10ml of methyl iodide was slowly added to the solution and the vial was shaken intermittently. Once all the methyl iodide was added, the vial was immediately sealed and heated to 120 ℃ under pressure and shaken for 3 hours ("hr"), the vial was cooled and unsealed, and the solvent and excess reagents were gently evaporated under a slow steady stream of nitrogen to obtain the methyl iodide complex of oltipraz as a red/brown solid. FTIR: c ═ S peaks 1197/1214 (relative intensity 2:8) and 431/419 (relative intensity 2: 8).
Example 6: process for making benzoyl chloride complexes of oltipraz (+) rotamers
20mg of crude oltipraz powder was added to a glass vial equipped with a rubber septum. To the vial was added 10ml of anhydrous chloroform at room temperature and the vial was placed on a shaking table until the resulting solution was clear. Using a syringe, 1ml of anhydrous benzoyl chloride was slowly added to the solution and the vial was shaken intermittently. Once all benzoyl chloride was added, the vial was immediately shaken for 15 minutes, the septum removed, and the solvent and excess reagents were gently evaporated under 10mm Hg vacuum to obtain benzoyl chloride complex of oltipraz as a red solid. FTIR: c ═ S peaks 1200/1213 (relative intensity 3:7) and 431/419 (relative intensity 3: 7).
Example 7: process for making diethyl dithiophosphate complexes of oltipraz (-) rotamers
22mg of the synthesized oltipraz powder was recrystallized using acetone-water and added to a glass round bottom flask equipped with a magnetic stir bar and a cold water condenser. To the flask was added 10ml of acetone at room temperature and while stirring was continued, 20mg of diethyl dithiophosphate ammonium salt was slowly added to the solution. Once all the diethyl dithiophosphate was added, the temperature was immediately raised and set to reflux for 3 hr. The released ammonia was captured from the top dilute hydrochloric acid trap of the condenser. After 3hr, the solvent was removed to yield the diethyl dithiophosphate/salt complex of oltipraz as an orange/red solid. FTIR: c ═ S peaks 1201/1220 (relative intensity 9:1) and 472/444 (relative intensity 8:2)
Example 8: process for making diethyl thiophosphate/salt complexes of oltipraz (-) rotamers
22mg of the synthesized oltipraz powder was recrystallized using acetone-water and added to a glass round bottom flask equipped with a magnetic stir bar and a cold water condenser. To the flask was added 10ml of acetone at room temperature and 19mg of diethyl thiophosphate/ammonium salt was slowly added to the solution while stirring was continued. Once all the diethyl thiophosphate/salt was added, the temperature was immediately raised and set to reflux for 3 hr. The released ammonia was captured from the top dilute hydrochloric acid trap of the condenser. After 3hr, the solvent was removed to yield the diethyl dithiophosphate/salt complex of oltipraz as an orange/red solid. FTIR: c ═ S peaks 1208/1221 (relative intensity 9:1) and 479/442 (relative intensity 9: 1).
Example 9: process for making dimethyl phosphate/salt complexes of the (-) rotamer of oltipraz
175mg of trimethyl phosphate/salt was placed in a round bottom flask equipped with a condenser and a stir bar together with 100mg of crude oltipraz (95% purity) and heated at 80 ℃ for 72 hr. After 72hr, the mixture was cooled to room temperature and washed five times with 50ml of toluene. 255g of orange/red dimethyl phosphate/salt complex of N-methyl oltipraz was collected from the lower layer and dried under vacuum at 60 ℃. FTIR: c ═ S peaks 1210/1227 (relative intensity 9:1) and 489/453 (relative intensity 9: 1).
Example 10: process for preparing isethionic acid complex of oltipraz (-) rotamer
22mg of crude oltipraz powder was added to a glass round bottom flask equipped with a magnetic stir bar and a cold water condenser. To the flask was added 10ml of acetone at room temperature and 30mg of ammonium isethionate was slowly added to the solution while stirring was continued. Once all of the ammonium isethionate salt was added, the temperature was immediately raised and set to reflux for 6 hr. The released ammonia was captured from the top dilute hydrochloric acid trap of the condenser. After 6hr, the reaction was brought to ph6.8 with a small amount of concentrated hydrochloric acid and the resulting mixture was evaporated to dryness under vacuum to give isethionic acid complex of oltipraz as a red/brown solid. FTIR: c ═ S peaks 1216/1228 (relative intensity 9:1) and 464/435 (relative intensity 9: 1).
Example 11: process for the manufacture of thiobenzoic acid complexes of the (-) rotamer of oltipraz
20mg of crude oltipraz powder was added to a glass vial equipped with a rubber septum. To the vial was added 10ml of anhydrous chloroform at room temperature and the vial was placed on a shaking table until the resulting solution was clear. Using a syringe, 1ml of ammonium thiobenzoate was slowly added to the solution and the vial was shaken intermittently. Once all the thiobenzoate ammonium salt was added, the temperature was immediately raised and set to reflux for 1 hr. The released ammonia was captured from the top dilute hydrochloric acid trap of the condenser. After 1 hour, the contents were removed under vacuum to yield the thiobenzoic acid complex of oltipraz as an orange solid. FTIR: c ═ S peaks 1210/1223 (relative intensity 9:1) and 474/438 (relative intensity 9:1)
Example 12: (+) and (-) rotamer pair H2O2Effect of induced ROS production in oxidatively stressed HGEPp cells
The formation of Reactive Oxygen Species (ROS) is indicative of oxidative stress. ROS accumulation coupled with increased oxidative stress is involved in the pathogenesis of many diseases, one of which is mucositis. See Sonis, A biological aproach to mucositis, J Support Oncol 2004; 2: 21-36; halliwell and Whiteman, Measuring reactive species and oxidative dam in vivo and in cell culture, how shouldou you do it and what do the results mean? British j.pharmacology, vol 142, stage 2, month 5 2004, 231-; and Iglesias-Bartolome et al, mTOR Inhibition predictions from Radiation-Induced Mucosites, Cell Stem Cell 11, 401-. Free radicals and other active species are constantly produced in the body and cause oxidative damage to biomolecules, a process that is limited by a variety of antioxidants and repair systems.
Example 7 of PCT/IB2017/001312 describes the use of HGEPp cells to determine the effect of oltipraz and formulated oltipraz crystals on intracellular ROSThis application was published as WO 2018/047013 to Applicant ST IP Holding AG. The formulated oltipraz crystals were typically prepared according to the process described in example 1 of PCT/IB2017/001312 (MHD less than about 350 nm). This same assay was performed on the rotamer compositions prepared in examples 1-11 above to determine their protection against primary human gingival epithelial cells (HGEPp) from hydrogen peroxide (H)2O2) Effects of induced oxidative damage.
Conditions are as follows:
as a result:
table 1 below shows the results of the above measurements on ST-617-DPI and the rotamer complexes of examples 1-11. The results of the ST-617-API (recrystallized oltipraz prepared according to the process disclosed in WO 2016207914) reported in Table 1 below are taken from FIG. 9 of PCT/IB2017/001312 (100 μ g/mL pretreatment).
TABLE 1
The decrease in ROS levels in the 100. mu.g/mL ST-617-DPI treated cell groups in the assay (122%) compared to the control (139%) was consistent with the results reported for 100. mu.g/mL in PCT/IB2017/001312 (123%), and other results were obtained for ST-617-DPI in this assay. The (+) rotamer complex provides a modest reduction in ROS, while the (-) rotamer complex provides a substantial reduction. The results of the determination of the (-) rotamer are statistically significant at 90% confidence level.
Example 13: differentially regulated oxidative stress-related genes in HGEPp cells following treatment with ST-617-DPI, ST-617-API, and rotamer complexes of examples 1-11
The Nrf2 system (discussed above) is considered a major cellular defense mechanism against oxidative damage by activating genes encoding phase II detoxification and antioxidant enzymes. One such oxidative damage is oral mucositis, which occurs during and after chemotherapy treatment, particularly head and neck cancer treatment. Relative expression of 84 stress genes was assessed using a human oxidative stress PCR array following pretreatment in HGEPp cells with 100ug/ml of a formulated oltipraz composition (MHD less than about 350nm), the rotameric complex described in examples 1-11 above, and a negative control (formulated oltipraz composition without oltipraz) generally prepared according to the process described in example 1 of PCT/IB 2017/001312.
Material
HGEPp cells were purchased from CellnTec Advanced Cell Systems AG. RNEasy Plus Micro kit was purchased from Qiagen n.v., USA. RNase-free DNase Set was purchased from Qiagen N.V., USA. RT2 simple first Strand kit (DNA Generator) and RT2Green fluor qPCR mastermix was purchased from Qiagen n.v., USA. Oxidative stress RT2 profiler PCR arrays (84 protection genes) were purchased from Qiagen n.v., USA. RT-PCR was performed using the iCycler PCR system from Bio-Rad Inc., USA.
Method
Cell culture:
pooled primary HGEPp cells were propagated in CnT-Prime epithelial cell culture medium supplied by CellnTec on 100mm petri dishes (petri dish) coated with 30mg/ml rat tail collagen type I (BD Biosciences) diluted in dalberg's phosphate-buffered saline (DPBS). HGEPp cells were grown at a density of 2.5X 104 cells/cm 2 in a Nunc cell size of 12X 60mmTMIn a cell culture dish.
Preparation of dosing solution:
formulated oltipraz crystals (ST-617-DPI) were prepared generally according to the procedure described in example 1 of PCT/IB 2017/001312. The recrystallized oltipraz is prepared according to the process disclosed in WO2016207914 ST-617-API. ST-617-DPI, ST-617-API and the rotameric complexes of examples 1-11 were dissolved in DMSO to reach a final concentration of 100. mu.g/ml. All dosing solutions contained 0.3% DMSO, which is well below the maximum percentage DMSO tolerance of HGEPp cells, i.e. 0.8%.
HGEPp cells were pretreated with ST-617-DPI and rotamer complex solutions:
1. the dish cell concentration was chosen to be 2.5X 105/ml to yield OD absorbance within the linear portion of the control curve.
2. Once HGEPp cells were cultured on the culture dish and ready (confluency had been reached), the medium was immediately removed and discarded. Cells were gently washed 2-3 times with DPBS and the last wash was removed and discarded.
3. HGEPp cell culture dishes were then pretreated with ST-617-API at a concentration of 100. mu.g/mL (in duplicate), ST-617-DPI and the rotameric complex for 24h at 37 ℃.
mRNA preparation from HGEPp cells:
mRNA was purified from HGEPp cells using RNeasy UCP Micro kit.
1. After discarding and washing the treatments, the cells were pelleted by centrifugation in a centrifuge tube at 1000RPM for 5 min. Carefully remove all supernatant by aspiration, ensure that all cell culture medium has been sufficiently removed.
2. Cells were disrupted by adding 350 μ l buffer RULT, carefully loosening the cell pellet from the tube and vortexing to mix well, and the mixture was homogenized by passing the lysate 5 times through a 20-gauge needle fitted to an RNase-free syringe.
3. 350 μ l of 70% ethanol was added to the lysate and it was mixed again by pipetting. (some precipitation was visible but not problematic for the assay)
4. Samples (including any pellet that may have formed) were transferred to RNeasy UCP MinElute spin columns in 2ml collection tubes and centrifuged at 10,000rpm for 15 s. The flow through was discarded.
5. Mu.l of buffer RUWT was added to the spin column and centrifuged at 10,000rpm for 15s to wash the membrane. The flow through was discarded.
6. Add 10. mu.l DNase I stock solution to 70. mu.l buffered RDD. Gently mix, add 80ul DNase I mixture directly to the spin column membrane and vortex for 15 min.
7. Mu.l buffer RUWT was added to the spin column and centrifuged at 10,000rpm for 15s to wash the spin column and the flow through was discarded.
8. Spin columns were placed in new 2ml collection tubes, 500. mu.l buffer RUPE was added and centrifuged at 10,000rpm for 15 s. The flow through was discarded.
9. The spin column was rewashed with 500 μ l 80% ethanol and placed in a new 2ml collection tube and centrifuged at full speed for 5min with the lid open to ensure removal of all ethanol.
10. The spin column was placed in a new 1.5ml collection tube, 14. mu.l of ultrapure water was added directly to the center of the spin column membrane, the lid was closed and centrifuged at full speed for 1min to elute the RNA. The dead volume of the spin column was 4. mu.l. Eluted with 16. mu.l of ultrapure water to give 20. mu.l (4. mu.g) of RNA eluate.
cDNA preparation for RT-PCR:
1. RNA samples from each of the above treatments were added to 40. mu.l buffer GE2(gDNA elimination buffer) and RNase-free H2O to make a final volume of 60. mu.l.
2. Incubate at 37 ℃ for 5min and immediately place on ice for 2 min.
3. 62 μ l of the BC5 reverse transcriptase mixture was added to each 60 μ l RNA sample to a final volume of 102 μ l.
4. Incubate at 42 ℃ for exactly 15 minutes and then immediately terminate the reaction by heating at 95 ℃ for 5 minutes. (keep on ice until qPCR (if needed)).
Human oxidative stress RT2 Profiler PCR array:
RT-PCR is a highly sensitive and reliable method for gene expression analysis. This assay was reliably used to analyze the expression levels of 84 genes associated with oxidative stress in HGEPp cells pretreated with 100. mu.g/ml of ST-617-API, ST-617-DPI and the rotameric complex for 24 h. The above cDNA was mixed with RT2 SYBR Green fluor Mastermix and aliquoted into wells of RT2 Profiler PCR array. RT-PCR was performed on an iCycler. Gene expression was compared using Ct values and results were calculated using the Δ Δ Ct method and normalized to the average expression level of 5 common genes (ACTB, B2M, GAPDH, HPRT and RPL 13A).
As a result:
table 2 below shows the results of the determination of ST-617-DPI and the rotamer complexes of examples 1 to 11. The results of ST-617-API (recrystallized oltipraz prepared according to the process disclosed in WO 2016207914) reported in table 2 below are from earlier assays performed as described above.
TABLE 2
The negative control did not show any changes in gene regulation. All rotamers showed a gene regulation response with (-) rotamer higher than (+) rotamer. The highest fold change between the two rotamers was for SOD1, GCLC, GCLM, and UCP 2. Notably, the GCLC and GCLM genes, which are genes for Glutathione (GSH) biosynthesis, show a clearly consistent difference between the (+) and (-) rotamers. These results correlate with the increase in serum GSH observed in clinical trials in patients receiving ST-617-DPI and experiencing low oral mucositis progression while undergoing radiation therapy for head and neck cancer. (-) rotamers showed a gene up-regulation response of ALOX12, SOD1, NQO1, GCLC and GSTP1 higher than ST-617-API. (-) rotamers showed a gene up-regulation response of SOD1, NQO1, GCLC, GSTP1 and GCLM higher than ST-617-DPI. Within the (-) rotamer, the dimethyl phosphate and isethionic acid complex (example 9 and example 10, respectively) showed significantly higher upregulation of SOD1, NQO1, GCLC, GSTP1 and GCLM than ST-617-DPI and ST-617-DPI. The (-) rotamer also showed differential down-regulation of UCP2 compared to ST-617-DPI and ST-617-API (mitochondrial uncoupling protein 2, which is involved in the control of mitochondrial-derived ROS).
Accordingly, the rotamer pharmaceutical compositions and methods of administering rotamer pharmaceutical compositions of the present disclosure may be used to treat any human or non-human animal patient to reduce intracellular Reactive Oxygen Species (ROS) and/or reduce oxidative stress, including in a patient undergoing a treatment that provides oxidative stress (e.g., chemotherapy or radiation therapy).
The rotamer pharmaceutical compositions and methods of administering the rotamer pharmaceutical compositions of the present disclosure may be used to treat any human or non-human animal patient to provide an antioxidant effect, including in patients undergoing treatment that provides oxidative stress (e.g., chemotherapy or radiation therapy). The rotamer pharmaceutical compositions and methods of administering rotamer pharmaceutical compositions of the present disclosure may also be used to slow the onset of, and/or reduce the severity of and/or shorten the duration of, oxidative damage (e.g., mucositis, including oral mucositis) in a patient, including in a patient undergoing treatment that provides oxidative damage (e.g., chemotherapy or radiation therapy).
Based on the above results, it is also believed that the (+) rotamer (including compositions in which a rotameric excess of the (+) rotamer is present) may upregulate genes associated with the Nrf2 pathway. Based on the above results, it is also believed that the (-) rotamer (including compositions in which a rotameric excess of the (-) rotamer is present) can upregulate genes associated with the Nrf2 pathway.
Some exemplary aspects are listed below.
1. A composition comprising a rotameric excess of the (+) rotamer of 4-methyl-5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione (oltipraz, see fig. 4A).
2. The composition of aspect 1, wherein the composition comprises one or more complexes of oltipraz (e.g., one or more salts of oltipraz) having a rotameric excess of the (+) rotamer.
3. The composition of aspect 2, wherein one or more oltipraz complexes comprise a salt derived from: adding a group (e.g., reversible addition of HCl, alkyl halide, or acyl halide) to the alpha nitrogen of the pyrazinyl ring of oltipraz produces an N-substituted pyrazinium ion and an associated anion, the alpha nitrogen resulting from adding the group to the pyrazinyl ring of oltipraz produces an N-substituted pyrazinium ion and an associated anion.
4. The composition of aspect 3, wherein the one or more oltipraz complexes comprise at least one oltipraz complex formed by addition to the alpha nitrogen of the pyrazinyl ring of oltipraz of one or more reactants selected from the group consisting of: alkyl halides, alkenyl halides, alkynyl halides, acyl halides, benzoyl halides, and combinations thereof (e.g., one or more reactants selected from the group consisting of HCl, methyl chloride, substituted or unsubstituted methyl, substituted or unsubstituted formyl, substituted or unsubstituted acetyl, substituted or unsubstituted benzoyl, substituted and unsubstituted butyl). Such oltipraz complexes may be formed by the reaction of oltipraz with, for example, formyl chloride, acetyl chloride, benzoyl chloride and n-butyl iodide, methyl iodide, and combinations thereof, respectively. In this aspect, the formyl, acetyl, benzoyl and butyl groups may be associated with the nitrogen atom at the 2-or 4-position of the pyrazine ring and may bear one or more substituents selected, for example, from halogen (e.g., F or Cl) and/or lower alkyl (C1-C6 alkyl).
5. The composition of any one of aspects 1-4, wherein the rotameric excess of the (+) rotamer is within a range selected from the group consisting of: 30% -40% excess, 40% -50% excess, 50% -60% excess and 60% -70% excess, 70% -80% excess and greater than 80% excess.
6. A composition comprising oltipraz complex, wherein the oltipraz complex has an excess of the (-) rotamer of 4-methyl-5- (pyrazin-2-yl) -3H-1,2-dithiole-3-thione (oltipraz).
7. The composition of aspect 6, wherein the composition comprises one or more oltipraz complexes having a rotameric excess of the (-) rotamer.
8. The composition of aspect 7, wherein at least one of the one or more oltipraz complexes comprises a direct bond between the alpha nitrogen and the electronegative atom of the pyrazinyl ring.
9. The composition of aspect 8, wherein the electronegative atom is selected from the group consisting of oxygen, sulfur, and phosphorus.
10. The composition of any one of embodiments 6-9, wherein the one or more oltipraz complexes comprise a hydroxymaleate oltipraz complex.
11. The composition of any one of aspects 6-9, wherein the one or more oltipraz complexes comprise one or more complexes formed by reacting oltipraz with one or more reactants selected from the group consisting of: carboxylic acids or salts thereof, phosphoric acids or salts thereof, alkylphosphates, arylphosphates, alkylsulfonic acids or salts thereof, arylsulfonic acids or salts thereof, substituted and unsubstituted thiocarboxylic acids or salts thereof, and combinations of one or more of the foregoing, for example, lactic acid, tartronic acid, isothionic acid, isoserine, 2-mercaptoethanesulfonic acid, taurine, propylphosphonic acid, 2-aminoethylphosphonic acid, diethyldithiophosphate, diethylthiophosphate, and dimethylphosphate.
12. The composition of any one of aspects 6-9, wherein the one or more oltipraz complexes comprise one or more complexes formed by reacting oltipraz with one or more reactants selected from the group consisting of: lactic acid, tartronic acid, isothionic acid, isoserine, 2-mercaptoethanesulfonic acid, taurine, propylphosphonic acid, 2-aminoethylphosphonic acid, diethyl dithiophosphate, diethyl thiophosphate, dimethyl phosphate, and combinations thereof.
13. The composition of any one of aspects 6-12, wherein the rotameric excess of (-) rotamer is within a range selected from the group consisting of: 30% -40% excess, 40% -50% excess, 50% -60% excess, 60% -65% excess, 60% -70% excess, 70% -80% excess, 80% -90% excess, and 90% -100% excess.
14. A pharmaceutical composition comprising at least one composition according to any one of aspects 1-13, wherein the composition is suitable for administration to a human.
15. The pharmaceutical composition according to aspect 14, wherein the composition is in a form suitable for topical administration.
16. The pharmaceutical composition according to aspect 15, wherein the composition is in a form suitable for rectal administration or administration by inhalation.
17. The pharmaceutical composition according to aspect 14, wherein the composition is in a form suitable for subcutaneous, intramuscular, intrasternal or intravenous injection.
18. The pharmaceutical composition of aspect 14, wherein the composition is in a form suitable for oral administration.
19. The pharmaceutical composition of aspect 18, wherein the composition is in the form of a powder, a pill, a tablet, or contained within a capsule.
20. The pharmaceutical composition of aspect 18, wherein the composition is in liquid form.
21. A process of treating a human or non-human animal patient in need thereof, the process comprising administering to the patient a pharmaceutical composition according to any one of aspects 13-20.
22. The process of aspect 21, wherein administering comprises oral administration.
23. The process of aspect 21, wherein administering comprises buccal administration
24. A process for preventing, treating, ameliorating, reducing the severity of and/or reducing the duration of mucositis in a human or non-human animal patient in need thereof, said process comprising orally administering a pharmaceutical composition according to any one of aspects 18 to 20.
25. A process for preventing, treating, ameliorating, reducing the severity of and/or reducing the duration of mucositis in a human or non-human animal patient in need thereof, said process comprising orally administering a pharmaceutical composition prepared according to any one of aspects 18 to 20.
26. A process for preventing, treating, ameliorating, reducing the severity of and/or reducing the duration of mucositis in a human or non-human animal patient in need thereof, said process comprising orally administering to the patient a pharmaceutical composition according to any one of aspects 18 to 20.
27. The process of any one of aspects 24-26, wherein the mucositis is oral mucositis.
28. The process of any one of aspects 24-26, wherein the mucositis is digestive tract mucositis.
29. A process for preventing, treating, ameliorating, reducing the severity and/or reducing the duration of mucositis or dermatitis in a human or non-human animal patient in need thereof, said process comprising topically administering a composition according to aspect 15.
30. A process for preventing, treating, ameliorating, reducing the severity of and/or reducing the duration of mucositis in a human or non-human animal patient in need thereof, said process comprising rectal administration of a composition according to aspect 16.
31. A process for preventing, treating, ameliorating, reducing the severity of and/or reducing the duration of mucositis in a human or non-human animal patient in need thereof, said process comprising administering by inhalation a composition according to aspect 16.
32. The process of any one of aspects 21-31, wherein the patient is undergoing radiation therapy.
33. A process for the manufacture of hydroxymaleic anhydride (-) rotamer of oltipraz, said process comprising the steps of:
(i) reacting oltipraz with (+) -diacetyl-L-tartaric anhydride in the presence of an anhydrous polar solvent to form a reaction mixture; and
(ii) acetic acid was added to the reaction mixture.