[001] ENZYME COMPOSITIONS, STEROID DERIVATIVES, ENZYME INHIBITORS, AND METHODS OF MAKING SAME FOR PHARMACEUTICAL APPLICATIONS

Background of the Disclosure

[002] Human Cytochrome P450 8B1 is the oxysterol 12α-hydroxylase enzyme that converts 7α-hydroxy-cholest-4-en-3-one to 7 a, 12 a -dihydroxycholest-4-en-3-one. This enzymatic activity ultimately results in the formation of cholic acid, the bile add with enhanced cholesterol absorption properties. Studies implicated this enzyme as a good drug target for nonalcoholic fatty liver disease and type 2 diabetes, but there are no selective inhibitors known for this enzyme and no structures to guide inhibitor development. As a sub-type of human cytochrome P450, the CYP8B1 active site may be compared with other similar P450 enzymes to identify features that may be useful in design of selective cytochrome P450 inhibitors across various sub-types.

Accordingly, while the present disclosure references compounds that inhibit P4508B1 activity, it is postulated within the scope of the present disclosure that the referenced compounds may exhibit inhibition activity across other P450 sub-types.

[003] Previously, a steroid that contains a C12-pyridine ring was designed and synthesized as a possible inhibitor for cytochrome P450 8B1. This inhibitor possessed a 3 β-hydroxy-D ⁵ - steroid system, which contrasts with the 3-keto-D ⁴ -steroid backbone of the physiological substrate. In order to determine if binding affinity changes when the ligand obtains a 3-keto-D ⁴ -steroid backbone in the A ring, two other variations of the originally designed inhibitor were synthesized: one with a 3-keto-D ⁴ steroid backbone and the other with a 3-hydroxy-D ⁴ -steroid backbone.

[004] None of the compounds elicited UV-Vis spectral changes of the Soret band when added to the enzyme, suggesting a lack of interaction between the ligand and the active site of the P450. However, out of the three compounds tested, only the steroid with the 3-keto-D ⁴ steroid backbone successfully crystallized with cytochrome P450 8B1. The ligand was not bound directly to the heme active site of P450 8B1, which was consistent with the binding experiments monitored with UV-Vis spectroscopy. Although the 12-pyridine compounds were not directly bound to the wild type P450 8B1 enzyme as monitored by UV-Vis absorbance assays and X-ray crystallography, these compounds served as tools to enhance our understanding of the structurefunction relationship of P450 enzymes.

[005] Cytochrome P4508B1 (P450 8B1 or CYP8B1) is the oxysterol 12α-hydroxylase enzyme responsible for converting its endogenous substrate, 7α-hydroxycholest-4-en-3-one, to 7α, 12α- dihydroxycholest-4-en-3-one (Figure 1, compounds 3 and 4, respectively). This activity results in the formation of cholic acid as shown in Figure 1. Chenodeoxycholic acid is the primary bile acid that is formed without P450 8B1 activity as shown in Figure 1. Mice lacking the gene that encodes for P450 8B1 resist weight gain and have improved glucose homeostasis through an increase of glucagon like peptide- 1 (GLP-1). Furthermore, knockdown led to regression in hepatic steatosis. Thus, the inhibition of P450 8B1 is a potential therapeutic strategy to treat obesity and cardiovascular diseases.

[006] Towards the efforts to develop a P450 8B1 inhibitor, a pyridine-containing steroid was synthesized as reported by Chung, E., et al., (2022) A synthesis of a rationally designed inhibitor of cytochrome P4508B1, a therapeutic target to treat obesity. Steroids 178, 108952, which was inspired based on the strategy for abiraterone, a prostate cancer drug, Barrie, S. E., et al, (1994) Pharmacology of novel steroidal inhibitors of cytochrome P45017a (17a- hydroxylase/C 17-20 lyase). J. Ster. Biochem. Mol. Biol. 50, 267-273. Abiraterone is a steroid inhibitor for P450 17A1, the 17α-hydroxylase enzyme that hydroxylates the 17-position of its steroid substrates, pregnenolone and progesterone. In order to target P450 17A1 activity, abiraterone bears (i) a 3β-hydroxy D ³ -steroid backbone, reminiscent of pregnenolone, the substrate for P450 17A1, and (ii) a pyridine at the 17-position, which contains the nitrogen lone pair that can coordinate to the iron active site of P450 17A1. Therefore, in order to access a rationally designed inhibitor for P450 8B1, a pyridine substituent was incorporated at the 12- position of a steroid molecule through a Suzuki cross coupling between 3-pyridylboronic acid and the vinyl iodide at the 12-position of the steroid. The vinyl iodide was synthesized from a dehydroepiandrosterone (DHEA) derivative, which contained a 3β-hydroxy-D ⁵ steroid backbone. [007] Previously, a P450 8B1 inhibitor with the 3β-hydroxy-D ⁵ steroid backbone, as shown in Figure 2 was used in mice, it was likely that this compound undergoes various transformations in vivo due to the presence of catalytically accommodating steroid metabolizing enzymes (3β- hydroxysteroid dehydrogenase and 170-hydroxysteroid dehydrogenase). For instance, abiraterone is delivered in vivo as a prodrug, abiraterone acetate, where its acetate at C3 is cleaved by esterases to its active form, abiraterone. In turn, abiraterone is oxidized and isomerized to the 3-keto-D ⁴ backbone by the action of 3β-hydroxysteroid dehydrogenase. Other downstream steroid metabolizing enzymes convert the steroid backbone of the abiraterone metabolite further (e.g. 5a-reductase) and have different biological activities from its parent compound.

[008] In the present disclosure, the steroid AB ring of the originally designed P4508B1 inhibitor, which possessed the 3β-hydroxy-D ⁵ AB ring system, was converted to the 3-keto-D ⁴ steroid backbone, as shown in Figure 2, compunds 7 and 8, in order to mimic the physiological substrate, 7α-hydroxycholest-4-en-3-one (Figure 1, compound 3). Furthermore, the 3-keto-D ⁴ steroid (compound 8) was reduced to the 3,17-dihydroxy D ⁴ steroid (compound 9) to determine if the different oxidation states at the 3- and 17-positions of the ligand potentially alter the interaction between the ligand and the enzyme.

[009] Only one of the three ligands bearing the 12-pyridine moiety in the steroid backbone (i.e. the 3-keto-D ⁴ compound 8), crystallized with P4508B1. Although it was determined that the 12- pyridyl 3-keto-D ⁴ steroid did not bind directly to the iron in the heme active site of P450 8B1, the structure of this complex was informative. The compound that crystallized with P4508B1, the 3-keto-D ⁴ compound 8, was the compound that resembles most closely to the endogenous substrate of P450 8B1 with its 3-ketone and double bond at the 4-5 position (i.e. compound 3).

Brief Description of the Figures

[010] Fig. 1 is an illustration of cholic acid and chenodeoxycholic acid biosynthesis from cholesterol.

[Oil] Fig. 2 is an illustration of the synthesis of 12-pyridine containing steroid analogs as putative inhibitors of P450 8B1.

[012] Fig. 3A is a three-dimensional structure of a 12-pyridine steroid analog showing distance calculation between C6 and the nitrogen atom and the furthest carbon atom of the pyridine heterocycle at Cl 2.

[013] Fig. 3B is a three dimensional structure of a 12-pyridine steroid analog showing distance calculation between the C12 position and the C5 position of the P450 8B1 substrate 7α- hydroxycholest-4-en-3 -one. [014] Fig. 4 is a primary sequence alignment between P450 8B1 (NP 004385.2), P450 8 Al (NP 00952.1), P4507A1 (NP 000771.2), P4507B1 (NP0048U.1), and P450 39A1 (NP 057677.2)), showing the conserved tryptophan residue. Y174, D211, W281, and R479 are marked with an asterisk (*).

[015] Fig. 5 A is a crystal structure illustration of P450 8 Al with an inhibitor showing the distance between the heme iron and the tryptophan-281 in the I-helix.

[016] Fig. 5B is a crystal structure illustration of P450 7A1 with cholestenone showing the distance between the heme iron and the tryptophan-284 in the I-helix.

[017] Fig. 6A is a NMR spectra of steroid analog compound 8.

[018] Fig. 6B is a NMR spectra of steroid analog compound 8.

[019] Fig. 7 A is a NMR spectra of steroid analog compound 9.

[020] Fig., 7B is a NMR spectra of steroid analog compound 9.

Detailed Description of the Disclosure

[021] The terminology used herein is for the purpose of describing example embodiments only and is not intended to be limiting. As used herein, the singular forms “a, ^: “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, integers, steps, operations, cells, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, cells, components, and/or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed.

[022] “Substantially” is intended to mean a quantity, property, or value that is present to a great or significant extent and less than, more than or equal to total. For example, “substantially vertical” may be less than, greater than, or equal to completely vertical.

[023] “About” is intended to mean a quantity, property, or value that is present at ±10%.

Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary methods of measuring and using such parameters. In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints given for the ranges.

[024] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the recited range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[025] References to “embodiment” or “variant”, e.g., “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) or variants) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment or variant, although they may. [026] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those maimers, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[027] Chemical nomenclature used herein is intended to be understood with reference to commonly employed nomenclature developed by the International Union of Pure and Applied Chemistry (IUPAC).

[028] Finally, steroid nomenclature used herein is intended to be understood with reference to the gonane structure having 17 carbon atoms arranged in four rings s conventionally denoted by the letters A, B, C and D. The gonane parent structure or steroid nucleus, is capable of being modified in practically unlimited manners by removal, replacement or addition of various moeties at various positions, including the saturation and unsaturation of bonds within the steroid nucleus. As used herein, all structures are intended to be disclosed in either or both the cis or trans isomers of the same.

[029] This detailed description of exemplary embodiments makes reference to the accompanying drawings, which show exemplary embodiments by way of illustration. While these exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosure, it should be understood that other embodiments may be realized and that logical changes and adaptations in design and construction may be made in accordance with this disclosure and the teachings herein without departing from the spirit and scope of the disclosure. Thus, the detailed description herein is presented for purposes of illustration only and not for purposes of limitation.

[030] Turning to Figure 1, there is illustrated the biosynthesis of cholic acid from cholesterol (compound 1). P450 8B1 incorporates the 12α-hydroxy group in its substrate, 7α-hydroxy- cholest-4-en-3-one (compound 3), to yield 7α,12α-dihydroxy-cholest-4-en-3-one (compound 4). Nine subsequent steps modify the A-ring and truncate the side chain to yield cholic acid (compound 5) (or chenodeoxycholic acid (compound 6) with no P450 8B1 activity) involve: (i) 3-oxo-5β-steroid 4-dehydrogenase, that reduces the C4-double bond), (ii) 3a-hydroxysteroid dehydrogenase to reduce the 3-ketone, (iii) P45027A1, (iv) solute carrier family member 27 member 5 (SLC27A5), (v) a-methylacyl-CoA racemase (AMCR), (vi) 3α,7α,12α-trihydroxy-5β- cholestanoyl-CoA 24-hydroxylase (ACOX2), (vii) 3-hydroxyacyl-CoA dehydrogenase (HSD17B4), (viii) sterol carrier protein 2 (SCP2), and (ix) acyl-CoA thioesterase, as described by Chevre, R., et al., (2018) Therapeutic modulation of the bile add pool bv knockdown protects against nonalcoholic fatty liver disease in mice. FASEB.J. 32, 3792-3802; Russell, D. W., et al., (1992) Bile Acid Biosynthesis. Biochemistry 31, 4737-4749, and/or Russell, D. W. (2003) The Enzymes, Regulation, and Genetics of Bile Acid Synthesis. Annu. Rev. Biochem. 72, 137-174.

Chemical Synthesis of Compounds

[031] A illustrated in Figure 2, the 3β-hydroxy-D ⁵ steroid with a 12-pyridine moiety (compound 7), was treated under Oppenauer conditions to yield the 3,17-diketone (compound 8). This resulting diketone was treated with NaBH ₄ to yield the 3,17-dihydroxy product (compound 9). These three compounds where then experimentally tested for P450 8B1 inhibition activity.

Experiments with 12-Pyridine Substituted Steroids with P4508B1

[032] When the ligands bearing a 12-pyridine moiety were added to purified P450 8B1, no shift in the Soret band was detected. Traditionally, inhibitors that containing nitrogen heteroatoms bind to P450 enzymes cause a type n binding difference spectra when monitoring the Soret band via UV-Vis spectroscopy. In contrast, addition of substrates to P450 enzymes usually result in type I binding difference spectra. Hence, the fact that none of the 12-pyridine bearing steroids caused any spectral changes when added to P450 8B1, suggested that none of these ligands directly bind to the heme active site of the enzyme.

[033] Crystal structure analysis was conducted and suggested that tryptophan-281 (W281) in the I-helix of P450 8B1 hovers about 7-8 A above the heme active site, limiting the size of the ligand that can access the active site of the enzyme. Therefore, since the distance between W281 and the iron in the heme active site of P450 8B1 and the distance between C6 and the furthest carbon in the pyridine is 9.2 A (Figure 3A), it is possible that the C12-pyridine steroids are too large to access the active site of P450 8B1. In contrast, the distance between C12 and C5 of the P450 8B1 substrate, 7α-hydroxycholest-4-en-3-one, is 5-6 A (Figure 3B).

[034] As shown in Figure 3 A, the tryptophan in the I-helix (W281), which is conserved in P450 8A1 (19), P450 7A1, P4507B1, and P45039A1, as reflected in the sequence alignment in Figure 4, plays a role in an interdomain interaction between the I-helix, D211 in the F-G loop, Y174 in the E-helix (Y169 in P450 8A1), and R479 beyond the L-helix (R480 in P450 8A1). [035] Furthermore, the distance between the tryptophan residue (W281) and the iron in the active site was about 7-8 A, which is smaller than the distance between the C5-position and the nitrogen atom in the pyridine heterocycle in the ligand at 9 A. Therefore, the wild type P450 8B1 should not be able to accommodate the space in the active site for the ligand in P450 8B1. The crystal structures of P450 8 Al and P4507A1 are available, and this conserved tryptophan residue is 7-9 A away from the iron in the heme active site, confirming that the key tryptophan may play a role in substrate recognition and binding to the active site.

[036] Although none of compounds 7, 8 or 9, yielded type II binding difference spectra with P450 8B1, inhibition assays suggested that the compounds have moderate inhibitory activity towards 12α-hydroxylation activity of 7α-hydroxycholest-4-en-3-one by P450 8B1.

W281F Mutation in P4508BI Has Enhanced Interaction "with I2-Pyridine Substituted Steroids

[037] To determine whether W281 of P450 8B1 plays a role in substrate (oxysterol) binding at the active site, a W281F site directed mutant was expressed and purified. When all three of the 12-pyridyl steroid compounds were added to the W281F mutant while monitoring by UV-Vis absorbance spectroscopy, a type 11 binding difference spectrum resulted. These observations confirm that the tryptophan plays a role in substrate binding above the heme active site. The W281F variant of P450 8B1 is not as active as the wild type, i.e. higher KM towards 12α- hydroxylation with the substrate, 7α-hydroxycholest-4-en-3-one). There were no alternative products identified by HPLC-UV analysis, scanning with 240 nm, when enzyme incubation extracts were analyzed. However, these observations do not rule out the possibility of other products formed by the P450 8B1 W281F variant, e.g. conjugation of the double bond could be lost if the enzyme epoxidized the double bond at C4 of compound 3.

[038] Three 12-pyridyl steroid compounds were synthesized and their interaction tested against human cytochrome P450 8B1. One of the compounds, bearing a 3-keto-D ⁴ steroid backbone (compound 8), was successfully crystallized with the enzyme. The compound was not bound to the heme active site, but the structure of the complex with the ligand allowed for a deeper understanding of how P450 8B1 binds with its substrate. Tryptophan-281 in the I-helix of P450 8B1, which interacted with D211 in the F-G loop and hovered 8 A, above the iron in the active site, was identified as a residue that may limit the length of the ligand for entry to the active site. Furthermore, binding studies with the W281F variant of P450 8B1 suggest that the ligand cannot reach the active site of the heme iron due to the W281 residue. Therefore, the 12-pyridyl steroid ligands are likely too long at about 9 A to be accommodated to bind in the active site of P450 8B1. Nevertheless, the 12-pyridine containing ligands are ample tools to investigate and understand the structure-function properties of human cytochrome P450 8B1 as a potential therapeutic target to treat obesity as well as other disorders mediated by P4508B1.

Synthesis of 12-(3 ’ -pyridyl) -androsta-4,1 l-dien-3, 17-dione (Compound 8)

[039] Aluminum isopropoxide (6.46 g, 31.7 mmol, 10 eq) and 1 -methyl -4-piperidone (7.78 ml, 63.3 mmol, 20 eq) were added to a 500 ml round bottom flask containing the C12-pyridine containing 3β-hydroxy-D ⁵ steroid 7 (1.15 g, 3.16 mmol, 1 eq) in toluene (250 ml). The reaction was refluxed using a Dean-Stark apparatus where toluene was removed in portions (3 x 20 ml). The volume of the reaction was never reduced below 150 ml. The progress of the reaction was monitored by TLC and NMR. After 24 h, a second portion of 1 -methyl -4-piperidone (10 ml, 86.6 mmol, 27 eq) was added to the reaction and refluxing continued for another 24 h. The reaction solution was diluted with ethyl acetate (200 ml). The resulting solution was washed with deionized water (3 x 200 ml). The aluminum isopropoxide suspension that crashed out during the workup was filtered off with a fritted filter funnel under vacuum. The organic layer was concentrated under reduced pressure to form a crude yellow oil. The crude oil was purified by column chromatography (50 % ethyl acetate hexanes — > 100 % ethyl acetate) to yield the 3-keto-D ⁴ -pyridine product 8 (1 g) with the l-methyl-4-piperidone as a major contaminant. This mixture was repurified by column chromatography (100 % ethyl acetate) to afford the 3-keto-D ⁴ -pyridine product 8 as a white fluffy solid (180 mg, 0.497 mmol, 16%); R/ 0.39 (100 % ethyl acetate); [α] _D ²⁰ + 239.3° [5.6 x 10 ⁴ in CHCh]; IR (ATR) 3459.13, 3025.93, 2927.84, 1736.33, 1663.04, 1447.04 cm" ¹ ; ^l H NMR (500 MHz, CDCh) 8 8.56 (s, 1H), 8.46 (s, 1H), 7.69 (d, J= 7.35 Hz, 1H), 7.25 (m, 1H), 5.83 (s, 1H), 5.55 (s, 1H), 2.61 - 2.34 (m, 6H), 2.26 - 1.90 (m, 6H), 1.88 - 1.79 (m, 1H), 1.70 (m, 1H), 1.38 - 1.29 (m, 1H), 1.27 (s, 3H), 1.22 (s, 3H); ¹³ C NMR (500 MHz, CDCh) 5 214.60, 199.08, 168.41, 149.18, 148.06, 143.06, 136.92, 136.26, 127.88, 125.77, 122.39, 56.42, 51.26, 49.11, 39.18, 36.57, 35.32, 33.74, 33.54, 33.18, 29.47, 20.39, 19.40, 17.96. The yield of this reaction was low due to the water workup step to remove the l-methyl-4-piperidone. Because both the pyridine product (8) and the 1 -methyl-4-piperidone reagent, which was in excess, are soluble in water, some of the desired product is lost in the aqueous layer. Furthermore, during the purification by silica gel column chromatography, the reagent (l-methyl-4-piperidone) co-elutes with the desired product. These mixtures were not isolated to move forward with pure compound in the next step. [040] Figures 6A and 6B are the respective NMR spectra for Compound 8.

Synthesis of 12-(3 ’-pyridyl)-androsta-4,l 1 -diene 3a,17b-diol (Compound 9)

[041] In a 50 ml round bottom flask containing the 3-keto-D ⁴ -steroid (8) (39 mg, 0.107 mmol, 1 eq) in methanol and THF solution (10 ml, 1:1 v/v) was added NaBH ₄ (67 mg, 1.77 mmol, 16.5 eq). The reaction was stirred for 20 minutes. The reaction was quenched with the addition of water (20 ml) and extracted with ethyl acetate (3 x 20 ml). The organic layers were combined and concentrated under reduced pressure to afford a crude white solid residue. The crude solid was purified by column chromatography (100 % ethyl acetate — > 10 % MeOH in CH2CI2) to afford the C3, C17 -diol product 9 as a white solid (17.3 mg, 0.047 mmol, 44 %); mp 194.6 - 198.5 °C; R/ 0.33 (100 % ethyl acetate); [a] _D ²⁰ - 2050° [2 x 10 ^-5 in CHCI3]; IR (ATR) 3282.61, 2918.36, 2849.79, 1588.03, 1435.00, 1259.65 cm ^-1 ; ¹ HNMR (500 MHz, CDCh) 88.55 (broad s, 2H), 7.71 (d, J= 10.15 Hz, 1H), 7.31 (m, 1H), 5.44 (s, 1H), 5.38 (s, 1H), 4.18 (m, 1H), 4.09 (m, 1H), 2.32 (apparent t, J= 11.81 Hz, 2H), 2.21 - 2.14 (m, 3H), 2.03 - 1.84 (m, 2H), 1.82 - 1.74 (m, 3H), 1.66 - 1.58 (m, 3H), 1.52 - 1.33 (m, 4H), 1.08 (s, 3H), 1.07 (s, 3H); ¹³ C NMR (500 MHz, CDCh) 8 148.86, 147.71, 146.18, 145.83, 136.33, 127.66, 125.55, 123.04, 76.80, 68.02, 57.73, 49.22, 48.21, 38.12, 35.06, 34.24, 32.89, 31.33, 30.94, 29.28, 22.10, 19.87, 15.66.

[042] Figures 7 A and 7B are the respective NMR spectra for Compound 9.

[043] While preferred embodiments have been described in detail, it should be apparent that modifications and variations thereto are possible, all of which fall within the true spirit and scope of the invention. With respect to the above description then, it is to be realized that the optimum dimensional relationships for the parts of the invention, to include variations in size, materials, shape, form, function and manner of operation, assembly and use, are deemed readily apparent to one skilled in the art, and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention.

[044] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" or the term "includes" or variations, thereof, or the term "having" or variations thereof will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In this regard, in construing the claim scope, an embodiment where one or more features is added to any of the claims is to be regarded as within the scope of the invention given that the essential features of the invention as claimed are included in such an embodiment.

[045] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within its spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[046] Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.

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