Patent Grant
7799937
This application claims priority from U.S. application Ser. No. 11/257,504 filed on Oct. 24, 2005, which claims priority from provisional application No. 60/621,747, filed Oct. 25, 2004.
This invention relates to novel metal coordinated complexes of biologically active molecules.
It is desirable to improve the properties of known, biologically active molecules by modifying their structures. The goal of such modifications is a molecule that is improved in some way, such as potency, stability, reduced side effects, or targeted delivery. This improvement is achieved without sacrificing the molecule's desirable properties. While this goal is easily stated, it is difficult to achieve in actual practice, as the effects of any particular modification is often highly unpredictable.
According to the current invention, the structure of known biologically active molecules is modified to result in new molecules known as metal coordinated complexes. These new molecules have unexpectedly superior properties. The metal coordinated complexes of the current invention include complexes of thyronine, tetracycline antibiotics, oxycodone and hydrocodone, and complexes of their derivatives.
Chelation is a critical component in the stabilization of a metal coordinated complex. For the s-block metals, this is particularly true for calcium and magnesium. For example, it can be seen that the log Keq of the acetic acid-magnesium complex is 0.47. With the incorporation of a single amino group on the molecule (i.e., glycine) the log Keq increased to 1.34. Magnesium typically prefers chelation with oxygen over nitrogen and this effect can be seen by comparing the log Keq of adenine (log Keq=2.08) with that of 6-hydroxypurine (log Keq=6.65). Magnesium forms particularly strong bonds with oxidized phosphorus, such as phosphates, as is revealed by comparing the log Keq of adenosine (log Keq=0.50) with that of adenosine-5′-monophosphoric acid (log Keq=1.80).
In general, zinc complexes are more stable then the comparable magnesium complexes. This is particularly true if the ligand bears nitrogen or sulfur. (This may not be the case for ligands with oxygen only and even less so if the ligand is a phosphate.) Using the glycine example above, the log Keq for the glycine-zinc complex is 4.85. The strength of the zinc sulfur bond versus the oxygen bond is manifest in the relative log Keq values for the zinc complexes of hydroxypropanoic acid (log Keq=0.86) and mercaptopropanoic acid (log Keq=6.43). Comparisons of log Keq values with other metals and ligands reveal that this chelation stabilization prevails in metal coordination chemistry.
Whereas it may not be required that chelation occur to form a stable metal coordinated complex with inherent covalency, and this is particularly true with the transition metals combined with nitrogenous ligands, in most cases it is a preferred embodiment of this invention that the active agent chelate with the metal, particularly if the metal is magnesium.
It is an embodiment of this invention that the active agents that make the best candidates for complexing with magnesium and calcium are those that have a proton on a heteroatom (i.e., oxygen, nitrogen or sulfur) with a pKa slightly greater than water or lower than water and have an additional heteroatom in close proximity to the first protonated heteroatom such that it can participate in the bonding, or otherwise chelate, with the metal. Drugs that have this arrangement of functional groups are most likely going to bond with a metal, where the resultant metal coordinated active agent will be stable enough in a biological system and survive hydrolysis therein, such that the performance of the active agent will be sufficiently modulated. This hydrolytic stability imparted by multidentate ligands is supported by the fact that they can lower the pKa's of the ligand such that even amides can be deprotonated with weak bases, such as triethylamine, in the presence of coordinating metals. Therefore, active agents with protons on heteroatoms, which normally would not be ionized in typical biological pH, can have the proton replaced with a covalently coordinated metal, where covalency is enhanced by the additional chelation from participating heteroatoms. It is a preferred embodiment of this invention that at least one of the heteroatoms on the active agent that will bind to magnesium or calcium be oxygen or sulfur. Magnesium forms unusually strong bonds with phosphates and phosphonates and, therefore, it is an additional embodiment of this invention that the active agent coordinated with magnesium is an organophosphate or organophosphonate compound.
It is an embodiment of this invention that the active agents that make the best candidates for complexing with zinc and the p-block metals are the same as those with the s-block metals with the additional flexibility that if the active agent has two nitrogens, a nitrogen and a mercaptan or two mercaptans in a proper chelation arrangement, then the presence of a proton on a heteroatom is not necessary to form a stable metal coordinated complex. It is a further embodiment of this invention that transition metals have further ligation flexibility in that chelation is even less required for their covalent coordination complexes if the ligands have at least one nitrogen or mercapto group.
The active agents which are embodied in this invention can be divided into chemical classes as shown in Table 1 (actually they may be divided into combinations of chemical classes to reflect the heterogenous chelation potential). The drugs listed in Table 1 are not intended to be an exhaustive list of all drugs that satisfy the embodiment of this invention but a representation of the chemical classes that exist in pharmaceuticals and that other pharmaceuticals that are of the same class listed in Table 1 or have arrangements of atoms that is satisfied by the embodiments of this invention are also claimed by this invention.
As illustrated in Table 1, a suitable biologically active moiety may have two functional heteroatom groups, each of which is capable of participating in the formation of a metal coordination bond. Further, as can bee seen from Table 1 the two functional groups may be in a spatial relationship to each other to permit chelation to the same metal atom by those coordination bonds. According to a preferred embodiment, the coordination bond forms a 4 to 8 atom ring encompassing the metal and the heteroatom of the functional groups and the ring usually does not include a trans double bond. The biologically active moiety and metal atom form a stable coordination complex as illustrated by (1) Table 1; (2) the examples set forth herein, such as Triiodothyronine (T3); (3) the figures, such as
The following discussion illustrates specific embodiments within the general principles discussed above.
Whereas it may not be required that chelation occur to form a stable metal coordinated complex with inherent covalency, and this is particularly true with the transition metals combined with nitrogenous ligands, in most cases it is a preferred embodiment of this invention that the biologically active moiety chelate with the metal, particularly if the metal is magnesium.
The biologically active moieties that have two functional heteroatom groups that are capable of participating in the formation of a stable metal coordination bond are embodied in this invention and include, but are not limited to the biologically active moieties set forth in Table 1. The biologically active moieties listed in Table 1 are not intended to be an exhaustive list of all biologically active moieties that satisfy the embodiment of this invention. The examples provided in Table 1 represent various groups of biologically active moieties that exist in pharmaceuticals or have arrangements of atoms that satisfy the embodiments of this invention.
The biologically active moieties listed in Table 1 have the attributes that make them a member of the group in which they are listed. They also have the functional groups that define that group and are in close enough proximity to each other to be able to chelate to a metal. Each group is defined by the specific combination of functional groups listed. For example, a guanide is a diamine but is also a specific kind of diamine. A guanide can actually be considered to be a triamine but since only two of the amino groups are necessary for chelating with a metal, the guanide and diamine are grouped together as a single chemical class. This is the same argument for the reason beta-diketone is grouped with beta-ketoalcohol, and diol is grouped with polyol.
Compounds that may be used in the embodiments of the invention, such as having two functional heteroatom groups and are capable of forming stable metal coordination bonds with a metal include, but are not limited to: Cladribine, Acetalzolamide, Eliprodil, (R,S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), Ifenprodil, (R)-4-oxo-5-phosphononorvaline (MDL 100453), Dihydroxyphenylglycine, (S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), Eglumegad (LY354740), (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG), Remacemide, Fingolimod, Teriflunomide, Laquinomod, Azathioprine, Clorazepate, Lorazepam, Temazepam, Rufinamide, Tiagabine, Progabide, Phenacemide, Lamotrigine, Ethoxzolamide, Zonisamide, Etoposide, Doxorubicin, Vorinostat (SAHA), Bicalutamide, 7-phenyl-2,4,6-hepta-trienal hydroxamic acid, Goserelin, Naltrexone, Fentanyl, Piritramide, Acadesine, Acarbose, Acebutolol, Acecarbromal, Acetylpheneturide, Acitretin, Adrafinil, Albendazole, Alexidine, Aliskiren, Alprenolol, Althiazide, Alvimopan, Ambuphylline, Amcamprosate, Amfenac, Amidephrine, Amidinomycin, Amiloride, 4-Amino-3-phenylbutyric acid, Aminophylline, Amlexanox, Amosulalol, Amprenavir, Arotinolol, Atorvastatin, Azidamfenicol, Baclofen, Balsalazide, Bambuterol, Bamethan, Befunolol, Benzthiazide, Betaxolol, Bevantolol, Bisantrene, Bitolterol, Brinzolamide, Bromfenac, Bromhexine, 5-Bromosalicylhydroxamic Acid, Bucillamine, Bucindolol, Bucumolol, Bufeniode, Bufetolol, Bufexamac, Buformin, Bufuralol, Bumadizon, Bunitrolol, Bupranolol, Buramate, Butanilicaine, Butazolamide, Butoctamide, Calcium N-Carbamoylaspartate, Capreomycin, Capuride, Carazolol, Carbazochrome Sodium Sulfonate, Carbimazole, Carisoprodol, Carmustine, Carteolol, Carticaine, Carubicin, Carvedilol, Catechin, Chloraminophenamide, Chlorguanide, Chlorphenesin Carbamate, Chlorproguanil, Chlorpropamide, Choline Alfoscerate, Cidofovir, Clodronic Acid, Clonixin, Cloranolol, Clorazepic Acid, Clorprenaline, Closantel, Cynarine, Dacarbazine, Delapril, Delavirdine, Denopamine, Diaziquone, 3,5-Dibromo-L-tyrosine, Diclofenac, Didanosine, Dideoxyadenosine, Digitalin, Digitoxin, Dioxethedrine, Dobutamine, Docarpamine, Docetaxel, Dorzolamide, Drotebanol, Droxidopa, Dyphylline, Ebrotidine, Ecabapide, Ecgonidine, Edatrexate, Eflornithine, Ellagic Acid, Endralazine, Enfenamic Acid, Entacapone, Epalrestat, Ephedrine, Epinephrine, Erdosteine, Ergotamine, Eritadenine, Esaprazole, Etanidazole, Ethylmethylthiambutene, Etidronic Acid, Etodolac, Exifone, Fenbendazole, Fendosal, Fenethylline, Fenoldopam, Fenoterol, Fenpentadiol, Fentiazac, Fepradinol, Flavopiridol, Fludrocortisone, Flufenamic Acid, Flunixin, Fluocortolone, Fluvastatin, Formoterol, Fosfosal, Gancliclovir, Gentisic Acid, Glafenine, Glibornuride, Gliclazide, Glimepiride, Glipizide, Gliquidone, Glisoxepid, Glyburide, Glybuthiazole, Guanabenz, Guanfacine, Hydrocortisone, Isoetharine, Isoflupredone, Isoladol, Lazabemide, Levobunolol, Lidamidine, Lopinavir, Lorazepam, Lormetazepani, Lotrafiban, Mefenamic Acid, Meglutol, Melagatran, Melphalan, Mepindolol, 6-Mercaptopurine, Metaproterenal, Methazolamide, Methisazone, Methocarbamol, Methoxamine, Methylergonovine, Metipranolol, Metoprolol, Midodrine, Mitoguazone, Mitoxantrone, Mivazerol, Mizoribine, Modafinil, Mopidamol, Moprolol, Moroxydine, Mycophenolate mofetil, Nadolol, Nadoxolol, Nalbuphine, Nalmefene, Naloxone, Nateglinide, Nebivolol, Nelfinavir, Nialamide, Nifenalol, Ninopterin, Nipradilol, Nitazoxanide, Nithiazide, Nolatrexed, Nordefrin, Norepinephrine, Norfenefrine, Norpseudoephedrine, Nylidrin, Octopamine, Omapatrilat, Onapristone, Orazamide, Osalmid, Orotic acid, Orthocaine, Oseltamivir, Oxazepam, Oxycinchophen, Oxyfedrine, Oxymorphone, Paclitaxel, Pamabrom, Pamidronic acid, Paramethasone, Penciclovir, Penicillamine, Perfosfamide, Phenazopyridine Hydrochloride, Pheneturide, Phenformin, Phenylephrine Hydrochloride, Phenyramidol, Phosphocreatine, Pindolol, Pipradrol, Pirarubicin, Pirbuterol, Piroxicam, Practolol, Prednylidene, Pregabalin, Prenalterol, Procaterol, Procodazole, Proglumide, Pronethalol, Propafenone, Propranolol, Protokylol, Pseudoephedrine, Quercetin, Quinocide, Raltitrexed, Rebamipide, Rebeccamycin, Reproterol, Ribavirin, Rilmazafone, Risedronic Acid, Ritodrine, Romurtide, Rufinamide, Salacetamide, Salicylamide, Sapropterin, Saquinavir, Sivelestat, Sotalol, Soterenol, Stepronin, Tafenoquine, Talinolol, Taltirelin, Tegaserod, Temazepam, Temozolomide, Tenoxicam, Terbutaline, Tertatolol, Theophylline, Thiamiprine, Thioguanine, Tiaprofenic Acid, Tilisolol, Tilarginine, Timolol, Timonacic, Tioclomarol, Tixocortol, Tocainide, Tolazamide, Tolbutamide, Tolcyclamide, Tolfenamic Acid, Toliprolol, Tolrestat, Torsemide, Tretoquinol, Triamcinolone, Tulobuterol, Ubenimex, Velnacrine, Vidarabine, Vigabatrin, Voglibose, Xamoterol, and Zoledronic Acid. Each compound listed above has other chemical properties that may require special conditions during the complexation reaction. The list of biologically active moieties and examples set forth herein provide a general protocol for the biologically active moieties listed and those related therein, but minor modifications to the applicable general protocols may be necessary for specific drugs, but are within the ordinary skill in the art.
Preferrably, compounds that may be used in the embodiments of the invention, such as having two functional heteroatom groups and are capable of forming stable metal coordination bonds with a metal include, but are not limited to: Acetalzolamide, Vorinostat, Aliskiren, Alvimopan, Bicalutamide, Baclofen, Balsalzide, Brinzolamide, Chlorproguanil, Diclofenac, Dorzolamide, Droxidopa, Clonixin, Ebrotidine, Enfenamic acid, Ethylmethylthambutene, Etodolac, Flufenamic acid, Fosfosal, Lazabemide, Mefenamic acid, 6-Mercaptopurine, Melagatran, Mycopheolate mofetil, Pregabalin, Quinocide, Rilmazafone, Tafenoquine, Tilarginine, Tolfenamic acid, Cidofovir, Didanosine, Dideoxyadenosine, Etidronic acid, Moroxydine, Nelfinavir, Pamidronic acid, Risedronic acid and Zoledronic acid.
For the present invention to be clearly understood and readily practiced, the present invention will be described in conjunction with the following figures, wherein:
Chemical bonds exist in three basic forms: ionic, covalent and coordination or the so-called Werner complexes, which are typically larger than inorganic metal salts. (It should be pointed out that Werner complexes are considered to have neutral ligands.) The differences between the three bond types can be attributed, in part, to the thermodynamic stability of the bond, particularly in solution. Conversely, the stability of a compound can be expressed as the propensity for the atoms of the molecule to separate or dissociate in solution.
The thermodynamic stability of a compound is expressed in terms of it free energy of formation according to equation 1:
ΔG=−RTlnK Equation 1
Where ΔG is the Gibbs free energy and indicates the thermodynamic stability of the compound. The more negative ΔG is the more stable the compound. R is the gas constant, T is the absolute temperature and K is the equilibrium constant. The equilibrium constant is expressed as a ratio of products over reactants. In the case of coordination compounds for the reaction:
M+xLMLx
K is expressed in equation 2:
K=[MLx]/[M][L]x Equation 2
Thus the increasing thermodynamic stability of a compound is directly related to the increasing value of the equilibrium constant.
In certain cases it is advantageous to express the equilibrium constant in terms the dissociation potential of a metal-ligand bond. The reaction is thus:
MLxM+xL
The dissociation constant, Kdiss is shown in equation 3:
Kdiss=[M][L]x/[MLx] Equation 3
Whereas it is commonly accepted that ionic bonds nearly completely dissociate in solution, most covalent bonds, do not dissociate at all. Thus, determining bond strength in solution, through measurement of the dissociation constant or the more commonly expressed parameter the equilibrium constant, is a method of discerning the bond type. For coordination compounds, which involve bonding between metals and ligands from Groups 15-17, the thermodynamic stabilities have not been firmly established.
Examination of the literature reveals that covalency of organometallic bonds can be determined from spectroscopic data (i.e. NMR and MS), ab initio molecular mechanics calculations or a combination of the two. In general, covalency is most likely to occur with transition metals, with nitrogen and sulfur ligand atoms (in preference to oxygen) and with increasing bond order or haptivity (designated “η”) from the ligand. Organometallic compounds with ligands that having multiple haptivities are described as chelates. Amongst the metals in groups 1 and 2, the so called s-block main group elements, only beryllium and magnesium are considered to be important chelate forming elements.
Recent research, using ab initio theoretical calculations, has further qualified the nature of the coordination bond in terms of the ionic vs. covalent nature of the ligand-metal bond. Pierloot applied the CASSCF (complete active-space self-consistent field) model to a series of Werner complexes to measure the degree of covalency of these organometallic complexes. Her general conclusions were that a trend exists wherein the static correlation energy, obtained from the CASSCF calculations, correlates well with covalency of the metal-ligand bond. She further concluded, that for the same metal, the metal-ligand covalency and related correlation effects increase in the following order of ligands:
F−<OH2<NH3<Cl−<Br−<I−.
This was in agreement with the nephelauxetic effect described by Jørgensen. The magnitude of this effect was directly correlated to the reduction of the interelectronic repulsion of a transition metal upon coordination in a ligand field. This reduction depended on the ligands and was expressed as the ratio of the Racah parameter, B, in the complex and in the free metal ion (β=Bcomplex/Bion). The reduction in B resulted from a decrease in electron-electron repulsion of the free metal ion after ligands were added to form the metal complex; a large reduction in B indicates a strong nephelauxetic effect. Thus ionic ligands, such as F−, give a small reduction in B and have larger β values. Based on spectroscopic measurements, ligands were ordered according to decreasing β values generating the nephelauxetic series:
F−>OH2>(NH2)2CO>NH3>H2NCH2CH2NH2≈(COO)22−≈(CO3)2−>NCS−>Cl−>CN−>Br−>N3−>I−>S2−≈(C2H2O)PS22−>diarsine
The correlation of the two series is supported by the similarities between the effects that both techniques described. In the former case, the CASSCF calculation gauges the contribution of the metal d-orbital to the metal-ligand bond. Racah parameter reduction by complex formation (Bcomplex) is caused by delocalization of the transition metal d-orbital electron cloud on the ligands, which is indicative of covalent bond formation.
Bonding between Li+ and Be2+ with Cp ligands is mostly ionic due to the low energy state of the contributing metal bond relative to the Cp bond. In addition, the ionic radius of these elements is too small to allow more than one Cp ligand to bond.
Theoretical calculations of magnesocene (
The reactivity of Cp2Mg and related Mg-Cp compounds was studied by Winter, et al., and they found that magnesium forms stable bonds with amidinate ligands. Perhaps most telling was the stability of [CpMg(η2-tBuC(N(2,6-iPr2C6H3))2)], which was sublimed unchanged with 80% recovery at 180° C./0.05 torr (
Whereas it is commonly accepted that transition metallocenes have strong covalent character, a legitimate argument that main group metallocenes have significant covalent bond elements has been made as well. The lack of d-orbital participation in the metal-ligand bonding may reduce the stability of the compound but does not preclude the notion that main group metals, particularly magnesium, can form bonds with ligands that are more covalent than ionic. It is generally known that the formation of 6-coordinate magnesium complexes upon their crystallization is due to sp3d2 hybridization. So it is conceivable that in certain situations even the d-orbitals of magnesium can participate in bonding of the coordination complex.
One can see a large difference in stability when comparing the equilibrium constants of a Werner complex with a metal halide. For example, log KMg-pyridine=2.08 and log KMgCl2≈−1.0. Keeping in mind that pyridine is a neutral ligand; this difference in log K can only be due to the covalency of the magnesium-pyridine bond vis-à-vis the ionic nature of the magnesium-chloride bond. Another example of a stable magnesium complex is seen with magnesium-salicylaldehyde (SA) complex, with a log KMg-SA2=6.80 (
The equilibrium constants of chelates are typically very large (e.g., log Keq for magnesium ethylenediamine-N,N′-disuccinate complex is 6.09) and may not reveal the extent of covalency between the neutral part of the ligand and the metal. However, it is the equilibrium constant that dictates the stability of any coordination compound and that is an important criterion for determining the nature of the chemical entity and how it will perform in particular applications. The existence of a covalent bond within the complex and its contribution to the stability of chelates can explain their very large log Keq and may also contribute to the rigidity of the molecular structure. It should be pointed out that, in many cases, covalency is the most important contributor to the stability of a coordination complex.
The magnesium porphyrin complexes or chelates are likely the most well known organomagnesium compounds; chlorophyll is a magnesium porphine. Phthalocyanine is a porphyrin representing the basic elements of that class of compound and is used extensively as a model system to study metal-porphyrin bonds. It has been determined that transition metals form complexes with phthalocyanine (
The synthesis, structure, stability and physical properties of metal-porphyrin complexes have been well studied. The structure and the physical properties of magnesium phthalocyanine have been further elucidated using a variety of techniques, most recently, near-IR absorption and X-ray crystallography. The recurring conclusion is that the magnesium-porphyrin chelate represents an extremely stable example of a metal coordinated compound.
Certain magnesium-ligand complexes are indeed covalent in nature and not ionic and thus are new compositions of matter and not merely new salt forms. Metal-organic ligand compounds, covalency in the nature of their bonds.
It is an embodiment of this invention that the formation of a coordination complex is favored when the ligand has direct bonding opportunity to the inner sphere of the metal, preferably magnesium. This is accomplished by using anhydrous magnesium and non-protic solvents (or if the solvent is protic it should be bulky). This concept is supported by the fact that the catalytic reactivity of a metal ion is reduced in its hydrated form. Complex formation in aqueous systems is a delicate balance between hydrogen bonds between ligand and water and the competition for binding sites on the metal by hydration and complexation capability of the ligand. It follows that complexation of a ligand with the inner sphere of metal is also reduced in aqueous systems. It further follows that the converse is true—that is, the rate of chelation or complexation of metals with ligands in non-aqueous systems is accelerated vis-à-vis aqueous systems.
A composition comprising an organic active agent bound to a metal as a stable metal-ligand coordination compound with inherent covalency is as a new molecular entity. In another preferred embodiment of the invention, the metal is selected from the main group elements. In yet a further embodiment of the invention, the metal is selected from the s-block elements. In a preferred embodiment of the invention, the metal is magnesium.
Furthermore, it is an embodiment of this invention that virtually any drug-magnesium complex with a Keq>1.0 has enough inherent stability to modulate the pharmacokinetics of dissolution, absorption, distribution, metabolism and excretion. Given that the dissociation constant of Mg(OH)2 is −11.5, it is not surprising to discover that most magnesium complexes are much more stable in alkaline conditions than in acid. Thus the stability of the drug-magnesium complex in the small intestines is likely to modulate the pharmacokinetics of drug absorption. For those metal-drug complexes that are acid labile, it is an embodiment of this invention that protection of the complex from the acidic milieu of the stomach be accomplished by a coating or encapsulation material that releases the complex upon entry into the small intestines. It is a further embodiment of the invention that the encapsulation agent is a ligand or group of ligands forming an outer coordination sphere.
Another important concept of this invention is that simple combinations of metal with ligands in solution do not always produce the same product. It is recognized that several, if not many, patents claim various salts as dependent claims without any support in the subject matter. This is accepted because the salt of an organic acid is easily prepared by treating it with a base and a metal salt where the expected product is the metal salt of the organic acid; a method known by anyone skilled in the art. However, when coordination chemistry contributes to the bonding between the organic acid and the metal, a variety of conditions, such as solvent, temperature and, perhaps most importantly, ligands attached to the metal, impact the structure and the stability of the coordination complex.
Additional ligands, other than the drug, can stabilize the metal-drug complex. For example the Keq of the glycine (G) magnesium bond is 1.34. If, however, salicylaldehyde is added to the complex, the equilibrium for the reaction
Mg2++SA−+G−Mg(SA)(G)
is 4.77. Clearly salicylaldehyde adds a stabilizing effect to magnesium glycine bond. It is an embodiment of this invention, that adjuvants, like salicylaldehyde are incorporated into the drug: metal complexes to impart beneficial physicochemical properties. It is a further embodiment of this invention that the benefit of adjuvants is to stabilize the drug: metal complex in certain environments, such as in aqueous solutions.
There are very few examples of coordination complexes with transition metals found in the Physician's Desk Reference (“PDR”) and include 1) insulin modified by zinc; 2) carboplatin contains platinum; 3) niferex is a polysaccharide-iron complex; 4) pyrithione zinc, used as the active ingredient in anti-dandruff shampoo. In addition, some nutritional supplements are described as complexes. Chromium picolinate is one example, where three picolinic acid groups are bound to a single Cr+3 in an octahedron (the nitrogens provide the three other binding sites). Whereas it is an embodiment of this invention that the metal is selected from the group representing transition metals in a more preferred embodiment of the invention the metal is selected from the s-block main group elements, groups 1 and 2. In a most preferred embodiment of the invention the metal is magnesium.
The patent literature cites some examples of novel magnesium coordinated drugs, which include: 1) Trilisate® a stable, solid choline magnesium salicylate composition mentioned above for treating arthritic pain; 2) magnesium salts of 2-descarboxy-2-(tetrazol-5-yl)-11-desoxy-15-substituted-omega-pentanorprostaglandins imparting greater tissue specificity and ease of purification and compounding into medicaments; 3) magnesium vanadate with insulinomimetic properties with utility in treating insulin resistance syndromes; 4) a crystalline magnesium-taurine compound for treatment of thrombotic or embolic stroke and prophylactic treatment of pre-eclampsia/eclampsia and acute cardiac conditions; 5) the magnesium omeprazole “salt” derivatives mentioned above to treat GERD.
The science of pharmaceuticals salts is a well studied area and selection of the salt form can impact a given pharmaceutical's performance. Examples of effects that the salt form can have on a drug include dissolution rate, solubility, organoleptic properties, stability, formulation effects, absorption modulation and pharmacokinetics. The periodical, Drug Delivery, published an article in October, 2003, citing three excipient applications using metals, presumably forming salts, to stabilize pharmaceutical agents. Human Growth Hormone is complexed with zinc to reduce its hydrophilicity and thereby slow the drug release; stabilization of proteins against the acidic environment produced by degradation of encapsulating polymers was accomplished by adding magnesium hydroxide to the formulation; zinc carbonate was used to stabilize vinca alkaloids from acid hydrolysis. Whereas these products clearly use metals to stabilize the pharmaceutically active agent, the latter two do not claim to have modified the structure of the active agent.
Further it is well established that by simply changing salt forms, the pharmacokinetics of absorption in the small intestines is significantly modulated. For example, the chemical structures of both the phosphate salt of tetracycline and tetracycline hydrochloride differ in that portion of the salt form which is not the pharmacophore and one would expect that the relative physical properties of each would not have a great influence on their relative bioavailabilities. But in fact, the phosphate salt is absorbed twice as much as the hydrochloride salt. Conversely, the bioavailability of the free acid of warfarin is nearly equivalent to its sodium salt, which is unexpected because the dissolution rate of the warfarin sodium tablet is 350 times faster than the tablet containing the free acid. If different salt forms can confer such changes in the kinetics of absorption, than a complex with an S-block main group element may have even a more pronounced effect on absorption.
It is known that the bioavailability of tetracycline antibiotics is mainly influenced by the physicochemical properties of their metal complexes that will most likely form in the GI tract. This is clearly an indication of drug:metal bond formation in vivo. The formation of a covalent bond between a drug and a metal in vivo is even a more reasonable expectation when the drug contains nitrogen and the metal is in Groups 10-12 (e.g., nickel, copper, zinc). It is an embodiment of this invention that modulation of a drug's performance is imparted by a formulation of the drug and the metal, which will facilitate formation of a stable complex between the drug and the metal.
It is an embodiment of this invention that the following benefits can be conferred upon a drug when complexed or coordinated with a metal:
1. Improved water solubility, which can equate to better bioavailability (see discussion below);
2. Enhanced lipophilicity for improved absorption through the cell membrane;
3. Locking the pharmacophore into a conformation for improved receptor binding;
4. Ameliorating formulation problems due to polymorphism (see discussion below);
5. Acid absorption properties for protection from degradation in acidic environments, such as the stomach;
6. The stability of the coordination complex may infer a delay in absorption of the active pharmacophore. This is important for drugs like liothyronine, where rapid absorption of the drug increases toxicity potential.
7. Bioadhesion properties for sustained absorption of the active pharmacophore;
8. Prevent abuse of narcotic analgesics by binding the pharmacophore of the narcotic through an organometallic complex to render the narcotic inert unless ingested.
Nature provides many examples of how transition metals are transported, stored and utilized. Perhaps the most well known example is hemoglobin, which is iron porphyrin. As stated earlier chlorophyll is a porphine structure surrounding magnesium. Some enzymes require metals in order for them to be active. That is the reason why trace metals, such as copper, zinc, chromium, etc. are important for proper nutrition. Even some antibodies have transition metals associated with them. The metal is required for enzyme activity due to the metal locking the peptide structure of the enzyme in a conformation through the formation of a coordination complex.
The concept of incorporating computer aided design of drugs has gained popularity in recent years. This technique, which has been referred to as in silico, has developed to the point that through the understanding of allosteric, coulombic and non-covalent interactions between the substrate and the receptor, lead drug candidates have been identified by computer modeling, before any material has been produced. It is an embodiment of this invention that by including metal coordination in the computer simulated molecule, new and improved lead compounds can be identified. It is a further embodiment of this invention that in silico derived lead compounds will have altered docking thermodynamics when the incorporation of a metal as a complex of the lead compound is included in the calculations. It is a yet further embodiment of this invention that compounds previously removed from consideration based on unsatisfactory in silico analyses will become important lead compounds when reanalyzed with the incorporation of a metal complex into the calculations. It is a preferred embodiment of this invention that the metal used for the revised in silico calculations as described above are selected from the main group elements. In a more preferred embodiment of this invention the metal is selected from the s-block main group elements. It is a preferred embodiment of this invention that the metal is magnesium.
In terms of direct application of the complex to a biological system, it is an embodiment of this invention that active agents that require ligand-receptor binding are imparted enhanced biological activity by virtue of the active agent's conformational structure being locked in place through complexation with a metal. The receptor can be membrane-associated, within the cytoplasm or circulating in the body. It is an embodiment of this invention that metals be incorporated into injectable drugs to lock the drug into a conformation that provides optimum interaction with its target receptor. It is a preferred embodiment of this invention that the metal be considered safe for injection. It is yet an even further embodiment of this invention that the metal be selected from the list of aluminum, bismuth, magnesium, calcium, iron or zinc. It is yet a further preferred embodiment of this invention that the active agent is selected from the list of injectable drugs, including, but not limited to, vaccines, antineoplastics, antidiabetic drugs, and antisense RNA or other metabolic modulators.
The metal coordination technology of this invention could also advance current research in vaccine design. For example, a new cancer vaccine being developed combines a lipoprotein adjuvant, a peptide antigen with a carbohydrate antigen specific for cancer cells. The three components of the vaccine construct are joined together covalently via linkers. This method of constructing the vaccine is common in bioconjugate chemistry. Metal coordination can be used as a scaffold to bind the different components of a bioconjugate such as Pegaptanib, whose combined components are an aptamer, polyethylene glycol and a lipid. It is an embodiment of this invention that the components of a bioconjugate can be combined in a single molecular entity by complexing each component to a central metal. It is a further embodiment of this invention that metal coordination serves as a general technique in bioconjugate chemistry.
Of particular note is the remarkable affinity that magnesium has for nucleic acids. With the advent of antisense RNA, interference RNA and aptamers as therapeutic agents it will be increasingly important to incorporate delivery technologies for these nucleic acid drugs. Some of the drug delivery techniques that are currently being investigated include pegylation, liposomes or anionic clays. Interestingly, a recent study released by Howard Hughes Medical Research Lab indicated that montmorillonite clay facilitated entry of RNA into lipid vesicles. There are a variety of clays that vary in the amount of alumina, silica, magnesia, iron and potassium. Thus, forming a magnesium complex of RNA may facilitate RNA's entry into vesicles, which are considered to be a laboratory model of cellular membranes.
The efficacy of the magnesium-nucleic acid complex can be evaluated vis-à-vis the nucleic acid alone using in silico techniques described above. Thus it becomes an important embodiment of this invention that nucleic acid drugs' efficacy is enhanced by their coordination with metals. It is a further embodiment of this invention that the nucleic acid be combined with a metal to form a coordination complex prior to administration. A significant portion of the complexes in simple combination of a metal salt with a nucleic acid in aqueous systems will be outer sphere coordinated ligands (
It is a further premise of this invention that inner sphere ligand formation is promoted by using anhydrous conditions to prepare the metal-ligand complex. It is an embodiment of this invention; therefore, that the metal ligand complex is prepared under anhydrous conditions and that reconstituting the complex in water will produce a coordination complex with greater covalency, greater stability, greater cell permeability and modulated biological performance relative to the complex prepared in water. This system is very amenable to incorporating adjuvants, such as polyarginine to enhance transfection efficiency, within the inner coordination sphere (
Perhaps the most important development in recent times in gene therapy was the discovery and advanced research on interference RNA (“iRNA”). Unlike antisense RNA, iRNA is recycled by the cell's biochemical machinery to further silence gene products encoded by mRNA. This results in increased efficiency of the gene silencing. The major problems associated with iRNA include their permeability into cells and their stability, particularly in the presence of nucleases.
These problems have been addressed by the incorporation of pulmonary surface active material (“SAM”), lipid or amine based transfection agents, electroporation, viral vectors or plasmid vectors. The latter technique is particularly interesting in that the plasmid vectors cause the siRNA to adopt a “hairpin” structure and these iRNA variants have been given the name of short hairpin RNA or shRNA. These shRNA molecules have enhanced silencing capacity. Moreover, there is a body of evidence that suggests that transfection agents are not necessarily required for the siRNA molecules to enter the cell. The recent discovery of pulmonary applications of siRNA and viroids are two reported phenomena wherein naked RNA can enter the cell and silence gene products. As a matter of fact, it is well known by scientists in this field that the secondary structure of the RNA does not seem to impact its gene silencing effects.
RNA is an oligonucleotide with multiple phosphate groups. Magnesium forms very strong bonds with phosphates and so RNA-Mg complexes are likely to have the magnesium atoms bound to the phosphate groups. By combining magnesium with RNA under anhydrous conditions, a covalent bond is formed, which, theoretically, would increase the lipophilicity of that portion of the RNA molecule. Furthermore, the magnesium center can bind multiple phosphate groups, theoretically, causing the formation of the hairpin structure mentioned above. This hairpin structure would not only manifest a lipophilic residue but would also provide greater resistance to attack from nucleases, which would lead to greater stability.
Since RNA would have other phosphate groups in excess of what is bound to magnesium, that portion of the RNA molecule would retain its water solubility. This novel form of RNA would have the desired amphiphilic properties that are important for mass transfer (hydrophilicity) and absorption (lipophilicity). For further discussion on this point see the “Improved solubility” section below.
A typical process would entail combining RNA with a magnesium salt in an anhydrous solvent. A suitable solvent may be DMSO or perhaps an ionic liquid. An advantage of ionic liquids is that recovery of the magnesium-RNA complex would merely entail adding the solution to an ionic liquid miscible non-solvent such as alcohol (or in some cases supercritical CO2 may work), where the desired product would precipitate out. The ionic liquid could then be recycled for the next reaction by distilling off the alcohol.
The above process would likely be applicable to any water soluble biologically active agent. Thus it is an embodiment of the invention that the biologically active agent is any saccharide, peptide or nucleotide. In a preferred embodiment of the invention the biologically active agent is a nucleotide. In a more preferred embodiment of the invention the biologically active agent is an antisense RNA, interference RNA or an aptamer. It is a preferred embodiment of the invention that the metal is selected from the main group elements. It is a further preferred embodiment of the invention that the metal is selected from the s-block main group elements. It is recognized that magnesium binds to nucleic acids more tightly than calcium, thus it is a most preferred embodiment of the invention that the metal is magnesium.
Improved Solubility/Permeability
In quantifying drug absorption it is useful to apply the term bioavailability. This is defined as the fraction (F) of the dose that reaches the systemic circulation. Thus, in the extreme cases, F=0 in drugs which are not absorbed at all in the GI tract while for drugs that are completely absorbed (and not metabolized by a first pass effect) F=1. The bioavailability can be calculated from the area under the curve (AUC) of the serum level vs. time plot. It depends on many factors and some of these factors differ between normal individuals. In terms of bioavailability, drugs have been classified into four categories according to the table below.
As can be seen a delicate balance between cell membrane permeability and solubility needs to be struck for a drug to become a viable candidate for further development. The reason for this is because physical properties that enhance solubility (i.e. hydrophilicity) are usually orthogonal to those properties enhancing permeability (i.e. hydrophobicity or lipophilicity).
The interaction between metals and tetracycline antibiotics has been shown to reduce the bioavailability of both the drug and the metal. As stated earlier, the bioavailability of tetracycline antibiotics are mainly influenced by the physicochemical properties of the metal complexes that prevail in the GI tract. Electric charge has the greatest impact on bioavailability since neutral species are more likely to readily absorb into the phospholipid membrane of the intestinal cells. A lipophilic metal coordinated complex should serve to allow greater bioavailability vis-à-vis metal salts of the drugs, which carry electric charges. Thus it is an embodiment of this invention that by administering lipophilic metal-antibiotic covalent complexes, physicochemical properties of the antibiotic can be controlled and, further, may prevent the metals in the GI tract to impact the dynamics of metal interaction with the drug and ultimately absorption. It is a further embodiment of this invention that the above stated principle is generally applicable to all drugs.
This technology can also be used to increase the lipophilicity of highly water soluble drugs, or the so called Class III drugs. In this case, the conversion of an ionic center, such as a phosphate or sulfate group, is converted to a covalent bond. This change in bonding between metal and ligand is known to decrease water solubility and increase organic solvent solubility or lipophilicity of the ligand.
If a drug is poorly soluble but is readily permeable one way its solubility can be enhanced is by covalently attaching water soluble entities such as amino acids or carbohydrates, to the drug. Alternatively, by forming a metal-ligand complex between the drug and an ionized metal center a new chemical entity is formed that now has inherent hydrophilicity imparted to it. It is an embodiment of this invention to bind the active agent to a transition metal or alkaline earth metal to form a new composition of matter that has improved solubility while retaining its permeability. Since the new metal ligand bond is covalent, it is preferable that the metal have additional ligands (e.g., amino acid) attached to it to counterbalance the lipophilic nature of the newly formed covalent metal center.
Due to the covalent nature the stability of the metal-active agent complex is retained up to transport to the water film coating of the brush border membrane. When the complex reaches the membrane the metal and the drug are separated by the lipids in the membrane accepting the lipophilic active agent and rejecting the hydrophilic metal. This is imparted through physicochemical action and, in contrast to the earlier methods of increasing solubilities of drugs, does not require enzymes.
Drugs are applied to the skin to elicit an effect to the 1) skin surface, 2) an effect within the stratum corneum, 3) an effect requiring deeper penetration into the epidermis and dermis or 4) a systemic effect through penetration to the vasculature. The aim of this research is to design a new transdermal drug delivery (TDD) system that will allow penetration of the drug through the epidermis or into the vasculature. The desired level of penetration will depend on the drug.
The stratum corneum provides an effective barrier and prevents water and chemicals from penetrating to the epidermis and beyond. It has been proposed that the structural organization of the lipids in the stratum corneum is an important factor in preventing fast transport of water and chemicals. This organization of lipids results in a liquid crystal morphology and penetration though this matrix is caused by destabilization of the liquid crystal through a disordering of the lipid hydrocarbon chains. This is the mechanism that has been proposed for the hydrotropes' ability to enhance penetration of topically applied drugs.
Some of the classes of chemicals that are used to enhance skin permeability include alcohols, alkyl methyl sulfoxides, pyrrolidones, surfactants (anionic, cationic and nonionic), and fatty acids and alcohols. In addition, laurocapram, urea, calcium thioglyclate, acetone and dimethyl-m-toluamide have been used to enhance skin penetration of specific bioactive reagents. Most of these drug vehicles' effect is by virtue of their hydrotropic properties. In chemical terms, many of them have a large dipole moment; that is they have a lipophilic portion and a hydrophilic portion. It is this large dipole moment which is a major contributing factor that causes these chemicals to disorder the lipids in the stratum corneum.
Many drugs do not intrinsically possess enough skin penetrative ability to be used topically. Thus, virtually every topically applied pharmaceutical requires a formulation that includes a vehicle or TDD enhancer in order to achieve the desired efficacy. Aside from the standard requirements of safety and efficacy to which all pharmaceuticals must comply, topically applied drugs need to be soluble and stable in the vehicle, the formulation must have content uniformity, the formulation must have proper viscosity and dispersion characteristics and must maximize patient compliance, which means it must not be uncomfortable to apply, have an unpleasant odor or cause skin irritation. Most notably, the lag time for the drug's penetration into the epidermis, which relies on its ability to partition from the vehicle into the stratum corneum, has presented significant obstacles during the development of TDD formulations. Previous reports show that this lag time can be anytime between minutes to several days. Thus, a major impediment of the development of TDD systems has been these additional considerations unique to this application and, historically, the development time for transdermal pharmaceuticals has often been viewed as exorbitant.
Enhanced transdermal permeability of a drug complex according to this invention relies on the stability of the complex coupled with its amphiphilic properties. Thus it is an embodiment that the formation of a covalent metal-drug bond converts the drug into an effective hydrotrope capable of enhancing TDD of the drug itself. It is a further embodiment of the invention that, if a TDD enhancer is still required, the metal will act as an anchor for the vehicle and the entire complex will behave as a single molecular entity. The advantage with this is that drug release from the complex no longer requires differential partition coefficients between the vehicle and the lipid matrix of the epidermis.
Due to its covalent nature, the stability of the metal-active agent complex is retained should be retained during transport through the stratum corneum. When the complex is in the epidermis the metal and the drug are separated by the lipids in the membrane accepting the lipophilic active agent and rejecting the hydrophilic metal. This is imparted through physicochemical action and, in contrast to the earlier methods of increasing solubilities of drugs, does not require enzymes.
Converting a drug to a metal coordination complex also facilitates entry into the eye. It has been shown that converting sulfonamides for treating intraocular pressure (IOP) to their metal coordination complexes increased their IOP reduction effect. It is believed that this is due, in part, to the increased presence of the sulfonamide in the eye and that this, in turn, is due to the right balance between lipo- and hydrosolubility of the metal coordinated complex. Drugs to treat eye diseases can be improved by converting them into a metal coordination complex according to this invention. This is very important to treat age-related macular degeneration (AMD), where the current therapies rely on injection of the drug behind the eye. An eye drop application of a drug to treat AMD greatly improves patient compliance; coordinating the AMD drug with a metal accomplish this.
Controlling Polymorphism
Polymorphism contributes a significant portion to the variability in dosages in part due to variation in solubility. Historically speaking, an inherent physical property of organometallic compounds is that stable crystalline forms are relatively easy to prepare. Thus it is a further embodiment of this invention that polymorphism is overcome by converting the active agent into a metal complex and subjecting the complex to recrystallization processes by methods commonly known by those skilled in the art. In so doing the active moiety is “locked” into a desired polymorph.
Modulating Drug Absorption
In recent times there has been a flurry of activity to improve drugs by modulating how the drugs are delivered. Drug delivery technology spans over all forms of administration from oral to injectable to implants to skin patches. Most of these technologies make use of an encapsulation technique or bead technology wherein the active ingredient is encapsulated or “trapped” inside a polymeric sphere. This polymeric sphere can exist as a micelle, as a self assembled molecular rod or ball or a coating around the active ingredient. The drug is released by solvation or swelling from the encapsulating agent as it circulates through the blood or traverses the gut. The main advantage of modulating the delivery of the drug is to extend its release, modulate the blood levels for improved safety or enhance its absorption for improved efficacy. Thus it is an embodiment of this invention that drug-metal complex release is modulated in vivo by physicochemical action on the complex itself.
In certain cases it may be beneficial to enhance the stability of the active agent-metal complex by encapsulating the drug-metal complex within a porphine, peptide or polymeric matrix. This is particularly true if the active agent does not contain the necessary elements for forming a stable complex with a metal, such as with the primary amines or alcohols mentioned above. It is a preferred embodiment of the invention that the matrix be a porphine derivative, modified, if necessary, to allow bonding of the active agent to the metal. It is an embodiment of this invention that drug-metal complex release is modulated in vivo by physicochemical action on the porphine, peptide or polymeric matrix. It is a further preferred embodiment of this invention that the matrix be a compound found naturally in the small intestines. In yet a further preferred embodiment of this invention the porphine matrix is bilirubin or a derivative thereof.
It is a further preferred embodiment of the invention that the encapsulating matrix is an amino acid or dipeptide, wherein amino acids or multiple dipeptides can be added to coordinate with or self assemble about the metal-ligand complex. Histidine is an ideal amino acid due to the strong metal binding capacity of the imidazole moiety in histidine. Arginine is another amino acid well suited for complexation with magnesium through amidinate ligation of the guanidine portion of peptide bound arginine (
The use of amino acids as secondary ligands on the metal is to stabilize the inner coordination sphere, create a hydrophobic shell about the inner sphere and thus preventing hydrolysis of the metal-drug bond. Thus, it is an embodiment of the invention that amino acids, dipeptides or oligopeptides act as secondary ligands or adjuvants on the metal-drug complex to stabilize the complex, particularly in aqueous systems. It is a preferred embodiment of the invention that the secondary ligand is a dipeptide. It is another preferred embodiment of the invention that the secondary ligand is an amino acid. In yet another preferred embodiment of the invention the amino acid is selected from the group histidine and arginine.
Organometallic complexes that have a free amino group (e.g. having an amino acid as part of the complex such as histidine) can initiate polymerization of an amino acid-NCA to form a polypeptide, conformationally protecting the organometallic complex. It is a further advantage of this technique to allow the amino acid NCA's to self assemble about the organometallic complex and then coacervating the polypeptide into its self assembled structure upon initiation of polymerization.
It is an embodiment of this invention to combine the encapsulation technology with the covalent technology to form an inner sphere covalent bond between the active agent and the transition metal, thus making a new composition of matter, and then encapsulating the complex with outer sphere coordination within a polymer matrix to provide a stable complex.
It is a further embodiment of the invention that the active agent only be released when the encapsulating matrix swells or is dissolved by water, oil, emulsions or biologic fluids such as gastric juices. It is an embodiment of the invention that the active agent cannot be released from the encapsulating matrix by virtue of the strong bond between the encapsulating agent and the active agent, such as what would occur with an antibody-antigen complex. In some cases it would be beneficial to have the release of the drug from the encapsulating agent be modulated by digestive enzymes. It is a preferred embodiment of the invention that the active agent is released from the encapsulating agent by its chemical breakdown by enzymes secreted in the intestines, within the cell membrane or circulating in the blood stream. It is preferred embodiment of the invention that the active agent is bound to aluminum, magnesium, calcium, iron, bismuth, silicon or zinc. In another most preferred embodiment of the invention the encapsulating agent is an antibody raised against the metal-ligand complex. In yet another embodiment of the invention the complex comprises an active agent-metal complex and the encapsulating agent is self-assembled from the combinations of amino acids, porphines, carbohydrates or mixtures thereof. In a most preferred embodiment of the invention the active agent-metal-encapsulating agent complex is a pharmaceutical.
In another embodiment of the invention, the coordination complex is a metal selected from all metals that can form such complexes, and the drug is selected from the group of all biologically or pharmacologically active agents. In a preferred embodiment of the invention the pharmacologically active agent requires a specific conformation for biological activity. The activity could be dependant on the active agent's ability to cross cell membranes and the coordinating metal provides the correct structure for membrane translocation of the active agent. In a preferred embodiment the pharmaceutically active agent is selected from the group consisting of small molecules, peptides, carbohydrates, DNA or RNA, the latter two being used in gene therapy, as aptamers or in antisense nucleotide therapeutic applications. In a preferred embodiment the metal is selected from the group consisting of aluminum, bismuth, calcium, magnesium, iron, silicon and zinc.
Bioadhesion Properties
There are a variety of ways that incorporating a magnesium or calcium ion into the molecular formula of a drug would infer bioadhesive properties to the drug. For example, it is known that magnesium and calcium are important for adhesive functions of integrins, thus it is reasonable to expect that a magnesium or calcium salt or complex of a drug in the intestinal tract would enhance bioadhesion of the drug to integrins expressed on the brush border membrane of the intestinal lining. And since bioadhesion translates into slower transit time in the gut, these complexes will confer sustained period of absorption in the gut. Therefore, it is an embodiment of this invention that sustained absorption of a drug will be enhanced by complexing the drug with magnesium or calcium. It is a further embodiment of the invention that said sustained release is conferred upon the magnesium or calcium drug complex by virtue of stronger bioadhesive properties.
Prevent Abuse of Narcotics
Narcotics are very effective analgesics but also can be very addictive. There have been many reports in the last few years describing the abuse of OxyContin by opiate addicts and recreational drug users. Typically the drug abuser will break the tablet matrix down mechanically or chemically, by adding water for example, thus making the full 12 hour dose available all at once. In addition, this type of abuse, which usually starts with oral administration, can often lead the abuser to snort or inject the concentrated narcotic.
Analysis of the structure of oxymorphone and oxycodone reveals that the molecules are ideal candidates for chelation with a metal. The β-hydroxyl at the 9-position and the nitrogen are positioned in such a way that complexing a metal between the two would form a highly thermodynamically favored 5-member ring. It is preferred that the 9-hydroxyl is deprotonated to form an anionic alkoxide (
Thus it is an embodiment of the invention that by virtue of the narcotic being complexed with a metal that the narcotic is released from the complex slowly through physicochemical action. This means that the narcotic is not available immediately or all at once. Furthermore, it is an embodiment of this invention that the metal-narcotic complex is unable to pass the blood brain barrier, rendering the narcotic ineffective until release from the complex has occurred. Since the kinetics of release is slow the amount of narcotic available for transport across the blood brain barrier at any one time is much less than the dose administered and so no euphoric effect is achieved. It is a further embodiment of the invention that the kinetics of narcotic release can be slowed even more by incorporating secondary ligands, encapsulating agents or a combination of both. In a preferred embodiment the metal is selected from the group consisting of aluminum, bismuth, calcium, magnesium, iron, silicon and zinc. In a more preferred embodiment of the invention the metal is selected from the main group elements. In an even more preferred embodiment of the invention the metal is selected from the s-block main group elements. In a most preferred embodiment of the invention the metal is magnesium.
Selection of Metals
Reference has been made to the preferred metals to be used in the coordination complexes. In pharmaceutical applications, the safety of the entire metal coordinated pharmaceutical needs to be considered when selecting the metal used in complexes of this invention. Although to practice this invention many metals can be used, it is a preferred embodiment of this invention that the metal be selected from a short list that would be generally regarded as safe (GRAS). One criterion for selecting the metal is to review the list of mineral supplements currently on the market and select the ones whose dosages would far exceed the dose likely to be included as a coordination complex with the drugs listed in Table 1. From the PDR for Non-prescription Drugs and Dietary Supplements a list of 7 metals (excluding alkaline metals, i.e. sodium, potassium, etc) with amounts greater than 2 mg/dose is shown in Table 2.
In a preferred embodiment of the invention the metal is selected from the group consisting of aluminum, bismuth, calcium, iron, magnesium, silicon and zinc. Whereas it is the embodiment of the invention that a new composition of matter is formed through the formation of a covalent bond between a pharmaceutical and any metal, including the lanthanides, actinides, the transition metals, and the main group metals (s- and p-block), it is a preferred embodiment of the invention that the metal be selected from the s-block main group elements. The reason for this is that the s-block elements are more likely to be GRAS and are more often used in OTC drug products and vitamin supplements than the transition metals or p-block main group elements (lanthanides or actinides are never used in OTC products). There are several reasons for selecting magnesium over the other s-block elements, such as calcium, which are:
Calcium shows larger variability with respect to coordination number with 8>7>6>9 in order of preference. Magnesium, being smaller than calcium, is almost exclusively octahedral, which simplifies the synthetic strategies and will more likely give a higher yield of a single product instead of a mixture of products.
Magnesium can form covalent bonds with chelating ligands more readily than the other s-block elements;
Magnesium forms a more stable bond with proteins and nucleic acids than calcium and thus provides enhanced stabilization of biologic pharmaceuticals.
Magnesium deficiency has been implicated in several disease states (e.g., cardiovascular related, migraine headaches, ADHD), and so from a prophylactic point of view magnesium may have significant benefit. For example, triptan magnesium may be an ideal candidate for this technology.
Calcium is absorbed in the intestines by an active transport mechanism, whereas magnesium is transported passively. Magnesium (as a salt) and Furosemide's intestinal transport were facilitated when both were co-administered orally. Thus Furosemide, a poorly absorbed drug, represents another compelling candidate for this technology.
It is a most preferred embodiment of this invention that the metal is magnesium.
Selection of Solvents
As stated earlier, the selection of solvent for the complexation reaction has an impact on the structure and stability of the metal coordinated compound. Magnesium forms strong bonds with water and the coordination sphere hydrated magnesium will have an impact on the kinetics of product formation as well as the structure and stability of the product. Because of the strong nitrogen-transition metal bond, in those cases where nitrogen containing ligands are reacted with transition metals, such as zinc, the presence of water in the reaction mixture will usually not have as strong an impact on the structure and stability of the metal coordinated product.
In some cases, depending on the ligand, the metal and the desired product water may be the solvent of choice. The majority of the products will dictate that an anhydrous organic solvent will be the best selection. Some suitable solvents include alcohol, acetone or THF. The most preferable solvent is DMSO because it is an excellent universal solvent that dissolves virtually every pharmaceutical or nutraceutical and also will dissolve most metal halides including magnesium chloride. This allows for single phase reactions. In addition, a stable metal coordinated pharmaceutical can be isolated by a process similar to coacervation, which typically will include simply adding a non-solvent to the reaction mixture.
DMSO can form complexes with metals, including magnesium, in situ, setting up the DMSO-metal complex to react with the drug ligand, thereby displacing the DMSO ligand at the metal center. DMSO can then serve as a transient protecting group in those reactions where adjuvants are to be included in the complex. This in-process reaction scheme is facilitated by the fact that the DMSO-metal complex cannot form outer coordination spheres like water does due to the lack of hydrogen bonding between the DMSO ligands. This makes the metal center easily accessible by incoming ligands. Depending on the metal coordinated complex formed, the final product may or may not retain DMSO as a ligand. If DMSO is attached to the ligand, it is unlikely that a situation will exist such that the dosing of DMSO will ever reach anywhere close to toxic levels.
It is the premise of this invention that by merely adding a metal salt to an aqueous solution of a biologically active ligand the coordination complex formed is not the same as if the combination of the reagents were done under anhydrous conditions. Furthermore, by reconstituting the dried coordination complexes in the same aqueous environment the structure of the two complexes would be different. To that end, several coordination complexes can be prepared with FDA approved pharmaceuticals, demonstrating that the complexes are stable. The products will be characterized as completely as possible and bioavailability studies will be conducted. The metal coordinated complexes can be prepared in water and organic solvents. It is expected that, in many cases, the respective products will have differences in stability, structure or biological activity.
Selection of Drugs
Complexes of almost any drug that can form a stable complex with a metal is enabled by this invention. The drugs selected for examples below represent a cross section of chemical and therapeutic classes as shown in Table 2.
Small Molecule Discussions
Synthesis
Other drug-magnesium complexes that may be important include Triptan-Mg due to the importance of magnesium for headache relief and Oxycodone-Mg because of the importance of abuse resistant narcotics.
Typically an anhydrous metal halide (iodide, bromide or chloride) is added to a mixture of the drug and KOtBu in tBuOH/DMSO. Alternatively, the metal halide can be added to the drug plus a solution of a tertiary amine (e.g. triethylamine) in DMSO. Yet another option is where the metal halide can be added to the drug plus KH in THF. The metals of choice are magnesium and zinc and the halide of choice is chloride. Zinc chloride is soluble in DMSO, acetone or ethanol and are the solvents of choice for zinc complexation, particularly with nitrogen containing ligands.
The product is isolated by precipitation, is separated from the liquid by suction filtration or centrifugation, washed and then dried under high vacuum to remove the last traces of moisture. The drug:metal complex may form a hydrate and all of the water may not be removable under high vacuum. Alternatively, the added water may not displace the remaining DMSO ligands on the metal formed in situ. Consequently, the product may be a drug:metal:DMSO complex.
Certain drugs, where their dissociation constants are high when bound to magnesium, favor complexation with DMSO. Formation of ternary complexes in situ would further stabilize the complexes and would retain their molecular integrity during the process of absorption after oral administration. It is for this reason that for most drugs when reacted with magnesium halide, DMSO is the solvent of choice.
As a comparator for the complexation reactions in the examples (excluding the acycloguanosine-Mg and T3-Zn examples), the reaction is repeated except water is included in the reaction medium as described in the examples below. The reaction is worked up and dried as before.
Adding water to the T3-Mg complexation reaction clearly had an impact on the isolated product. The T3-Mg compounds prepared in DMSO alone, showed line broadening in the aliphatic region only, with sharp aromatic peaks, in its 1H NMR spectrum (
Tetracycline has β-diketone and β-ketophenol functionalities and will form stable complexes with magnesium. Adding water to the reaction in DMSO has very little effect on the solubility, the 1H NMR spectra or the metal content of the respective products. In fact the 1H NMR spectrum of the product isolated from a reaction done in water alone does not differ significantly from the product isolated from DMSO alone or in a 5:1 DMSO:water mixture. There is a trend of lower magnesium content with higher water content in the reaction solvent, but that may be due, primarily, to extent of hydration in the product.
Since zinc forms a very stable bond with nitrogen containing compounds, water does not interfere with complexation between zinc and the ligand but may impact the complex structure. The product resulting from reacting a metal and a drug in anhydrous DMSO typically yielded well characterized coordination complexes. For those compounds that were either not well defined structurally (i.e. HCTZ-Zinc) or were somewhat unstable (i.e., dimethylbiguanide-zinc complex), a zinc coordination complex was isolable and was, at least partially, characterized. The same complexes prepared in the presence of water, had higher solubility in polar solvents. The difference in solubilities of the products from the respective methods of preparation clearly established a difference in the products themselves. It is believed that the zinc products prepared in aqueous solvent systems produced ionic salts, outer coordination complexes, hydrated complexes or combinations thereof. This appeared to be the case in the dimethylbiguanide-zinc complex prepared in 5:1 DMSO:water mixture, where the 1H NMR spectrum revealed resonances at 2.85 and 2.80 ppm, which correspond to the N-methyl groups and indicate free (or ionic) and complexed dimethylbiguanide, respectively.
Characterization
Each product synthesized were characterized by NMR, MS (either TOF or FAB) and ICP. The NMR spectrum confirms the integrity of the sample and shows that a metal is complexed by the presence of line broadening, peak shifts or multiple resonances.
Metal content for most of the complexes prepared in anhydrous DMSO were consistent with a complex of two drugs bound to a metal. In addition, the metal content remained constant from batch to batch. The complexes of Dimethylbiguanide had variable metal content depending on the method of isolation and never had consistent drug:metal ratios. The metal content was determined by the ICP analysis and based on that data, in conjunction with the NMR and MS, the ratio of drug to metal can be calculated.
The 1H NMR spectra of the T3 complexes showed some line broadening and upfield shifting in the aliphatic region indicative of complex formation with the amino acid portion of the molecule. This can be seen by comparing the region between 2.5 ppm and 3.5 ppm in the 1H NMR spectra of, bis(T3)bis(DMSO)Mg, T3, free acid and bis(T3)Zn, which are shown in
The 1H NMR spectrum of the dimethylbiguanide complexes showed large upfield shifts of the —NH resonances indicative of complex formation with the nitrogen atoms. In addition, a 0.05 ppm upfield shift of N-dimethyl groups was observed in the dimethylbiguanide-zinc complex spectrum (
The 1H NMR spectrum of the minocycline and tetracycline complexes resembled polymeric structures with very large line broadening and manifestation of many new resonances throughout the entire spectrum. To demonstrate that these spectra were due to dynamic isomeric mixtures and not decomposition or polymerization, 12 N HCl was added to the NMR samples of tetracycline and its magnesium complex and the spectra retaken.
The 1H NMR spectrum of the hydrochlorothiazide complexes also resembled polymeric structures with the same kind of line broadening and new nondescript resonances seen in the spectra of the antibiotic-metal complexes.
Spectral data on the acycloguanosine-magnesium complex data showed that a complex was formed. Comparison of the 1H NMR spectra of acycloguanosine with that of its magnesium complex (
The mass spectrum revealing a significant presence of the coordination complex is an important indicator of the stability of the complex. Thus, molecular ions were found for bis(T3)Mg, bis(T3)Zn, bis(minocyclinato)Mg, bis(tetracyclinato)Mg, and bis(acycloguanosinato)Mg. Two molecular ions with zinc isotope patterns were observed in the MALDI spectrum of hydrochlorothiazide-zinc complex. It is not known, at this time, a structure corresponding to those masses. Dimethylbiguanide-zinc complex did not have a zinc containing molecular ion in its MALDI spectrum. This is believed to be due to the compound's instability.
FTIR studies may be used to determine whether, for a particular complex that has been found, the ligand bonding atom and if the complex is coordinated with DMSO, water, or not solvated at all.
Stability
Equilibrium constants for the coordination complexes made have been estimated from literature precedents of similar compounds. For example, the equilibrium constant, log Keq for T3-Zn is estimated to be between 4 and 5 based on another amino acid zinc complex, phenylalanine-zinc. Likewise, the log Keq for dimethylbiguanide-zinc is estimated to be between 5 and 7. The log Keq for hydrochlorothiazide-zinc is difficult to estimate from literature values. Stability constants for tetracycline-magnesium in water at various pH values have been reported and the expected log Keq for tetracycline-magnesium is between 4 and 5. The log Keq for acycloguanosine-magnesium is estimated to be 1.6. The log Keq for T3-Mg is difficult to estimate due to the lack of a good comparator but the log Keq for glycine, which like T3 is also an amino acid, is 1.34. Due to T3's much greater hydrophobicity relative to glycine, the log Keq for T3-Mg is expected to be much larger than 1.34.
Another indicator of drug-metal or drug-metal-adjuvant stability is their binding constants, which is related to Keq but further shows the stepwise stability for multi-dentate ligands. The cumulative binding constant, βn, for the maximum binding between the metal and the ligand is given by Equation 4.
This difference can be seen in the measured log Keq for phenylalanine-zinc and its approximated β2 value of 8.5. This β2 value may also more closely reflect the stability of the T3-Zn complex.
There are several ways in which binding constants of metal-drug complexes in different environments can be estimated. Optical absorption spectroscopy, NMR spectroscopy, mass spectrometry, reaction kinetics, potentiometry and chromatography are several such methods.
Partition Coefficient and Distribution Coefficient
The partition coefficient is a constant and is defined as the ratio of concentration of a neutral compound in aqueous phase to the concentration in an immiscible organic phase, as shown in Equation 5.
Partition Coefficient, P=[Organic]/[Aqueous] Equation 5
In practice the Log P, defined in Equation 6, will vary according to the conditions under which it is measured especially pH since at a low pH bases will be ionized and at a high pH acids will be ionized.
Log P=log 10(Partition Coefficient) Equation 6
Thus, a Log P=1 means 10:1 Organic:Aqueous, a Log P=0 means 1:1 Organic:Aqueous and a Log P=−1 means 1:10 Organic:Aqueous.
Naturally, ionized compounds will partition preferentially into the aqueous phase, thereby lowering their log P. For neutral molecules that are bases they will remain neutral when the pH is greater than 2 units above its pKa and for neutral acids when the pH is 2 units below its pKa.
The choice of partitioning solvent will also have an impact on log P. Most log P measurements will use the octanol:water system. Ion pairing effects impact the log P measurements and should be accounted for, especially with metal coordinated compounds such as those embodied in this invention.
In terms of pharmaceutical applications the following guidelines have been used in determining the method of administration, formulation and dosage forms:
And within the realm of orally administered drugs these guidelines have been used:
The distribution coefficient (D) is the ratio of unionized compound in the organic phase to the total amount of compound in the aqueous phase given by Equation 7.
D=[Unionised](o)/[Unionised](aq)+[Ionised](aq) Equation 7
Log D is the log distribution coefficient at a particular pH (Equation 8). This is not constant and will vary according to the protogenic nature of the molecule. Log D at pH 7.4 is often quoted to give an indication of the lipophilicity of a drug at the pH of blood plasma.
Log D=log10(Distribution Coefficient) Equation 8
LogD is related to LogP and the pKa by the following equations:
Log D(pH)=log P−log [1+10(pH−PKa)] for acids Equation 9
Log D(pH)=log P−log [1+10(pKa−PH)] for bases Equation 10
So, when the pH is adjusted such that ionization is minimized, the log D will be nearly equivalent to the log P. Under those conditions, then, log D is a reliable indicator of the bioavailability of a drug in a particular application. In terms of a metal coordinated drug, increases in log D of the drug-metal complex relative to the reference drug will not only indicate an increase in lipophilicity but will also demonstrate its stability in water, as well. The log D's at pH 7.4 for tetracycline, bis(tetracyclinato)magnesium, triiodothyronine and bis(triiodothyroninato)zinc were determined and are shown in Table 4 along with their pKa's.
These results run counter to what the prior art teaches; that is combining the anion of a neutral compound with a metal salt should produce a compound with less lipophilicity and a reduced log D. Perhaps more interestingly, by application of the technology in this invention, tetracycline was theoretically transformed from an injectable drug (log D<0) to an oral drug (log D>0) and T3 was transformed from an oral drug (log D<3) to a transdermal drug (log D>3). This latter manifestation has important implications in increasing the safety of T3 products by administering the drug in a slow release transdermal depot.
Bioavailability Studies
Overview: A preliminary study in a rat model to observe the effects that coordinating a metal with a drug will have on the absorbance of the reference drug was conducted. The reference drug selected for this particularly study was triiodothyronine (T3), which is the active ingredient in Cytomel and Thyrolar. Both Cytomel and Thyrolar are currently used to treat hypothyroidism. Cytomel has also been indicated in the treatment of certain psychological disorders.
Study Design: T3-Zinc and T3-Magnesium were tested for bioavailability, relative to the reference drug over a 5 hour time period. The three compounds were formulated, separately, into gelatin capsules, with a total dose of 108±12 μg/kg administered. In order to avoid acid degradation of the test compounds in the rat stomach, metal oxides were added to the formulation. Another T3 control was included, where zinc oxide was added to T3, free acid. Each of the formulated gelatin capsules were orally administered directly to the esophagus of respective rats and blood samples were collected at pre-dosing and at 0.5, 1, 2, 2.5, 5 hours after dosing. Serum triiodothyronine levels were analyzed by an independent laboratory, using an industry standard assay method.
Results: The data shown in the
Conclusions: The data clearly indicate that complexing a metal with T3 increased the absorbance of orally administered T3 nearly two fold over T3 alone. It is believed that the T3, free acid complexed with the zinc oxide prior to absorption of T3 in the animals fed the formulation of T3 and zinc oxide.
This study represents a significant advance in drug delivery technology and that coordinating metals with pharmaceuticals can be applied to and improve the performance of drugs with bioavailability limitations.
Large Molecule Protocols
Synthesis
Double stranded iRNA of a defined size were prepared according to the standard protocol described in the Examples section. The iRNA was then reacted with magnesium or zinc under anhydrous conditions or in the presence of water as described in the Examples section.
The biological activity of iRNA can be modulated in various ways by complexing it with other metals such as calcium, zinc, cobalt and manganese. In addition, combinations of multiple metals, such as including Cu or Ag33 to facilitate binding of the purine/pyrimidine groups along with the phosphate groups can improve the transfection efficiency and stability of iRNA.
Characterization
The metal:RNA products were tested for changes in ionic/covalent behavior using Isoelectric focusing (IEF) gel following the standard protocol described in the Examples section. The advantage of the IEF gel is that it provides an inexpensive tool by which to monitor changes in the charge distribution on the target RNA molecule. Data from the IEF studies were represented as ΔpKa for each iRNA complex prepared.
Conclusion of IEF Experiments
The presence of an RNA molecule from the anhydrous DMSO reactions with a different isoelectric point (pKa) indicates the presence a new RNA molecule (
FTIR Analysis of DMSO-Magnesium Complex
In order to determine which atom of DMSO binds to magnesium and FTIR spectrum was collected of a DMSO-magnesium complex. The FTIR spectrum showed an extra stretch at 954 cm−1, which is indicative of an S═O—Mg stretch. FTIR of T3 complexes were examined for the presence of S═O—Mg, C═O and N—H stretches.
The following examples illustrate compounds having lower water solubility and carboxylic acid functionalities when complexed with a metal. DMSO is a preferred solvent, to which a strong or weak base is added, such as potassium t-butoxide or potassium carbonate, followed by the salt of the complexing metal, preferably the chloride salt. The preparation also includes water precipitation with or without concentrating in vacuo. This method can be applied to the amido acid and thio acid.
Preparation of Bis(triiodothyroninato)-bis(dimethylsulfoxide)magnesium
Triiodothyronine or T3 (218 mg) was dissolved in 4 mL of anhydrous DMSO, after which 0.34 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Magnesium chloride (16 mg) was added and the solution stirred overnight. The solution was poured into 10 mL of deionized water to precipitate the product, which was suction filtered and air dried. After an overnight drying under high vacuum the yield was 164 mg of a light beige powder. The product structure was characterized by 1H NMR, FAB-MS and ICP. 1H NMR (DMSO): δ 7.83 (s), 7.05 (d), 6.82 (d), 6.62 (dd), 3.26 (bm), 3.15 (bd), 2.71 (bm), 2.54 (s). The presence of the broad multiplets in the side chain region indicates that as the site for magnesium binding (see
Preparation of Bis(triiodothyroninato)zinc
Triiodothyronine or T3 (192 mg) was dissolved in 4 mL of anhydrous DMSO, after which 0.30 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Zinc chloride in diethyl ether (0.16 mL of 1M solution) was added and the solution stirred overnight. The solution was poured into 10 mL of deionized water to precipitate the product, which was suction filtered and air dried. After an overnight drying under high vacuum the yield was 140 mg of a light beige powder. The product structure was characterized by 1H NMR, FAB-MS and ICP. 1H NMR (DMSO): δ 7.82 (s), 7.02 (d), 6.80 (d), 6.61 (dd), 3.49 (bm), 3.22 (bm), 2.67 (bm). The presence of the broad multiplets in the side chain region indicates that as the site for zinc binding (
Preparation of Magnesium Triiodothyronine Complex in the Presence of Water.
Triiodothyronine or T3 (188 mg) was dissolved in 3.5 mL of anhydrous DMSO, after which 0.29 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Magnesium chloride (16 mg) in 0.5 mL of water was added and the solution stirred overnight. The solution was poured into 10 mL of deionized water to precipitate the product, which was suction filtered and air dried. After an overnight drying under high vacuum the yield was 188 mg of a light beige powder. The product structure was characterized by 1H NMR, FAB-MS and ICP. 1H NMR (DMSO): δ 7.81 (bs), 7.02 (bs), 6.81 (bd), 6.59 (dd). The other resonances were hiding behind the solvent and water peaks. The sample formed a cloudy suspension in DMSO. FAB-MS: Molecular ion at 652 indicative of protonated T3 with no bound Mg. FTIR (neat): cm−1 1633 (N—H stretch), 1535 (C═O stretch), 1012 (S═O stretch), 949 (S═O—Mg stretch). The strength of the DMSO related stretches relative to the T3 related stretches indicated a mixture of (DMSO)xMg cation and T3 anion. Magnesium analysis (ICP): Expected 1.8%; Found 0.96%. Potassium analysis (ICP): Found 0.23%. From the ICP data it appears that this product is a mixture of the magnesium salt, the potassium salt and the zwitterion.
The following examples illustrate the use as starting materials compounds that may be unstable. Examples of such compounds are the β-diketone, α-diketone, ketophenol, α-ketoalcohol, or β-ketoalcohol chemical classes. DMSO is a preferred solvent, to which a strong base is added, such as potassium t-butoxide, followed by the salt of the complexing metal, preferably the chloride salt. The preparation also includes water precipitation with or without concentrating in vacuo.
Preparation of Bis(minocyclinato)magnesium
Minocycline (104 mg) was dissolved in 3 mL of anhydrous DMSO, after which 0.44 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Magnesium chloride (11 mg) was added and the solution stirred overnight. The solution was poured into 10 mL of deionized water to precipitate the product, which was suction filtered and air dried. After an overnight drying under high vacuum the yield was 52 mg of a deep yellow powder. The product structure could not be characterized by 1H NMR, possibly due to the different permutations of bidentate complex forms possible with anionic Minocycline and magnesium. The product was characterized, then, by FAB-MS and ICP. FAB-MS: Molecular ion at 937.4 indicative of bis(minocyclinato)magnesium. Magnesium analysis (ICP): Expected 2.68%; Found 2.61%. This confirms that there are two Minocycline molecules per magnesium atom, as represented by a likely structure shown in
Preparation of Bis(tetracyclinato)magnesium
Tetracycline (89 mg) was dissolved in 0.5 mL of anhydrous DMSO, after which 0.2 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Magnesium chloride (11 mg) was added and the solution stirred for 3 hours. The solution was concentrated in vacuo at 30° C., after which 0.5 mL of deionized water was added and the mixture triturated and transferred to a 2 mL microcentrifuge tube. The product was separated from the liquid by centrifuging at 8,000 rpm for 6 minutes and the supernatant was decanted. The pellet was washed by adding 0.5 mL of water, vortex mixed, centrifuged and the supernatant decanted. The washing procedure was repeated. After an overnight drying under high vacuum the yield was 72 mg of a deep yellow powder. The 1H NMR spectrum (
Preparation of Magnesium Tetracycline Complex in Water
Tetracycline (89 mg) was dissolved in 1.5 mL of water, after which 0.2 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Magnesium chloride (0.11 mL of 1 M solution) was added and the solution stirred for 3 hours. The resultant precipitant was separated from the water by centrifuging at 8,000 rpm for 6 minutes and the supernatant was decanted. The pellet was washed by adding 1 mL of water, vortex mixed, centrifuged and the supernatant decanted. After an overnight drying under high vacuum the yield was 65 mg of a deep yellow powder. The 1H NMR spectrum very closely resembled the complex prepared in anhydrous DMSO. Magnesium analysis (ICP): Expected 2.67%; Found 2.42%. It appears that performing the complexation in water versus under anhydrous conditions has a minor impact on the stability and structure of the tetracycline-magnesium complex.
The following examples illustrate the use as starting materials compounds that belong to any of the amine, diamine, guanide, diamide, certain purine, sulfonamide, and oligopeptide chemical class. DMSO is a preferred solvent, to which a strong base is added, such as potassium t-butoxide, followed by the salt of the complexing metal, preferably the chloride salt. The preparation also includes alcohol precipitation with or without concentrating in vacuo.
Preparation of Hydrochlorothiazide Zinc Complex
Hydrochlorothiazide (120 mg) was dissolved in 0.5 mL of anhydrous DMSO, after which 0.4 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Zinc chloride in diethyl ether (0.2 mL of 1M solution) was added and the solution stirred for 4 hours. The solution was concentrated in vacuo at 30° C., after which 0.5 mL of methanol was added and the mixture triturated and transferred to a 2 mL microcentrifuge tube. The product was separated from the liquid by centrifuging at 8,000 rpm for 6 minutes and the supernatant was decanted. The pellet was washed by adding 0.5 mL of methanol, vortex mixed, centrifuged and the supernatant decanted. The washing procedure was repeated. After an overnight drying under high vacuum the yield was 104 mg of a free flowing white powder. The product was apparently hygroscopic due to the powder turning gummy after a few minutes exposure to ambient air. The 1H NMR spectrum resembled a polymeric structure (
Reaction between Zinc and Hydrochlorothiazide in the Presence of Water
This procedure followed the analogous anhydrous preparation exactly except the zinc chloride was added to 100 μL of water and the ether was allowed to evaporate off prior to adding to the reaction mixture. No product was isolated because the entire mixture slowly dissolved in methanol during the precipitation step. This was putatively due to the formation of methanol soluble hydrated complexes.
Preparation of Dimethylbiguanide Zinc Complex
Dimethylbiguanide (66 mg) was dissolved in 1 mL of anhydrous DMSO, after which 0.88 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Zinc chloride in diethyl ether (0.22 mL of 1M solution) was added and the solution stirred for 3 hours. The solution was concentrated in vacuo at 35° C., after which 0.5 mL of ethanol was added and the mixture triturated and transferred to a 2 mL microcentrifuge tube. The product was separated from the liquid by centrifuging at 8,000 rpm for 6 minutes and the supernatant was decanted. The pellet was washed by adding 0.5 mL of ethanol, vortex mixed, centrifuged and the supernatant decanted. The washing procedure was repeated. After an overnight drying under high vacuum the yield was 57 mg of a free flowing white powder. 1H NMR (DMSO): δ 4.90 (s), 4.66 (s), 4.50 (s), 2.80 (s). The large upfield shifts of —NH protons relative to dimethylbiguanide indicate complexation with zinc. A small upfield shift of 0.05 ppm in the dimethyl groups was also observed in the dimethylbiguanide-zinc complex.
Preparation of Zinc Dimethylbiguanide Complex in the Presence of Water
This procedure followed the analogous anhydrous preparation exactly except the zinc chloride was added to 100 μL of water and the ether was allowed to evaporate prior to adding to the reaction mixture. Work up and drying was followed in the same manner to yield 47 mg of a free flowing white powder. The compound was sparingly soluble in DMSO resulting in low signal to noise ratio in the NMR. 1H NMR (DMSO): δ 4.90 (bs), 4.66 (bs), 4.50 (bs), 2.85 (s), 2.80 (s). The singlet at 2.85 ppm indicates the presence of dimethylbiguanide that is not complexed with zinc. Judging from the integration of the two peaks at 2.85 and 2.80 ppm, the ratio of free dimethylbiguanide to the zinc complex is about 1:1.
Preparation of Zinc Dimethylbiguanide Complex in Water
Dimethylbiguanide (33 mg) was dissolved in 1 mL of water, after which 0.44 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Zinc chloride in diethyl ether (0.22 mL of 1M solution) was added and the solution stirred for 5 hours. The resultant precipitant was separated from the liquid by centrifuging at 8,000 rpm for 6 minutes and the supernatant was decanted. The pellet was washed by adding 1 mL of water, vortex mixed, centrifuged and the supernatant decanted. After an overnight drying under high vacuum the yield was 20 mg of a free flowing white powder which contained no organic material by 1H NMR. The isolated product was zinc salts in various hydrated forms.
Preparation of Bis(acycloguanosinato)magnesium
Acycloguanosine (45 mg) was dissolved in 0.5 mL of anhydrous DMSO, after which 0.2 mL of 1 M potassium t-butoxide in t-butanol was added and the solution stirred for 10 minutes. Magnesium chloride (11 mg) was added and the solution stirred for 4 hours. The solution was concentrated in vacuo at 30° C., after which 0.5 mL of methanol was added and the mixture triturated and transferred to a 2 mL microcentrifuge tube. The product was separated from the liquid by centrifuging at 8,000 rpm for 6 minutes and the supernatant was decanted. The pellet was washed by adding 0.5 mL of methanol, vortex mixed, centrifuged and the supernatant decanted. The washing procedure was repeated. After an overnight drying under high vacuum the yield was 15 mg of a beige coarse powder. The product structure was characterized by 1H NMR, MALDI-ES and ICP. 1H NMR (DMSO): δ 7.70 (s), 6.75 (bs), 5.30 (s), 4.64 (bs), 3.42 (s). MALDI-ES: Molecular ion at 473.1 is suggestive of bis(acycloguanosine)magnesium. Other molecular ions at 666, 702, 893 and 1065 have not been assigned to a particular structure. Magnesium analysis (ICP): Expected 5.14%; Found 4.16%. FTIR data did not indicate presence of a DMSO ligand. From a combination of the absence of the amide proton at 10.6 ppm, the ICP analysis and the MALDI-ES analysis, the structure for bis(acycloguanosinato)magnesium is shown in
The following examples illustrate the use as starting materials compounds that belong to any of the thio azole, azole amine, azole amide, ureide or certain purine chemical classes. DMAC is a preferred solvent, to which the base and the complexing metal are added as a single reagent (e.g., Magnesium tert-butoxide). The mixture is heated, preferably under reflux conditions and followed by removal of the solvent in vacuo.
Preparation of Magnesium Azathioprine Complex
Azathioprine (azaH, 50 mg, 0.18 mmol) was dissolved in dimethylacetamide (5 mL) and Mg(OBut) (20.2 mg, 0.09 mmol, 90% purity) was added. The mixture was heated to reflux for 2.5 hours. The reaction turned orange. The reaction was cooled to room temperature and concentrated under reduced pressure to afford a solid material. Analysis by 1H NMR indicated formation of the chelate. 1H NMR (DMSO-d6) d 8.35 (s, 1H), 8.20 (s, 1H), 8.18 (s, 1H). The 1H NMR (DMSO-d6) of azaH is: d 8.60 (s, 1H, H2), 8.57 (s, 1H, H8), 8.26 (s, 1H, H11).
The structure of azathioprine is as follows:
The following examples illustrate the use as starting materials compounds that are very stable and have two functional groups, both of which can be deprotonated, such as acid alcohols, diols, polyols (e.g. polysaccharides), dicarboxylic acids and certain β-ketoacids. DMAC is a preferred solvent, to which a strong base is added, such as potassium t-butoxide, followed by the salt of the complexing metal, preferably the chloride salt, or the metal oxide. The mixture is then heated, preferably under reflux conditions. The preparation also includes concentrating the reaction mixture in vacuo, extracting the metal coordinated product with alcohol, and removing the alcohol in vacuo.
(Salicylato)zinc
Sodium salicylate (1.60 g, 10 mmol) was dissolved in DMAC (100 mL) and a solution of potassium tert-butoxide in THF (10 mL, 10 mmol, 1 N) was added dropwise with stirring at room temperature. To this suspension was added ZnO (408 mg, 5.0 mmol) and the mixture was heated at reflux for 18 hours. The reaction was cooled to room temperature and the precipitate was filtered thru a fritted funnel (medium frit). The solid was washed with EtOH causing the suspension to pass thru the frit leaving an insoluble solid behind. The EtOH fraction was collected and concentrated to afford product. (Salicylato)zinc. 1H NMR (DMSO-d6) d 7.46 (br s, 1H), 6.84 (brs, 1H), 6.36 (br s, 1H), 6.14 (br s, 1H).
A sample of the zinc salt of salicylic acid was prepared by reacting sodium salicylate (2 equiv) with ZnCl2 (1 equiv) in H2O at room temperature overnight. The precipitate was filtered, washed with H2O and EtOH to afford zinc salicylate. 1H NMR (DMSO-d6) d 7.76 (dd, 1H), 7.30 (m, 1H), 6.76 (m, 2H), in agreement with literature values
The structure of (Salicylato)zinc is as follows:
The following examples illustrate the use as starting materials compounds that belong to any of the thio acid, amino acid and certain β-ketoacid chemical classes. DMF is a preferred solvent, to which the base and the complexing metal are added as a single reagent (e.g., Magnesium tert-butoxide). The mixture is stirred at room temperature and followed by removal of the solvent in vacuo.
Preparation of Bis(furosemide)magnesium
Furosemide was obtained from Kemprotec, Ltd. Solvents and reagents were obtained from Sigma-Aldrich and were used without further purification. Oasis Hydrophilic-Lipophilic Balanced (HLB) reversed phase Solid Phase Extraction (SPE) cartridges were obtained from Waters Corporation. NMR spectra were obtained on a Varian 400 MHz instrument. Elemental analyses were performed at Galbraith Laboratories. Inductively Coupled Plasma (ICP) spectrometry was performed at REI Consultants. Reagents for the calorimetric titration of Mg were obtained from Hach Corporation.
To a 25 mL round bottom flask equipped with magnetic stirrer was added 1.00 g (3.02 mmol) of furosemide and 10 mL of anhydrous DMF. To this solution was added 277 mg (1.51 mmol) of Mg(t-BuO)2. The suspension was stirred for 18 h at room temperature. During the course of the reaction, the suspension turned opaque. Solvent was removed under reduced pressure at 50° C. and the yellow oil was further dried in a vacuum oven (50° C., 3 torr) overnight. The semi-solid material was dissolved in 3 mL MeOH. Water (57 mL) was added and this solution was applied to an Oasis HLB solid phase extraction cartridge (35 cc, 6 g). The column was eluted sequentially with MeOH/H2O fractions of increasing solvent strength. The fractions were concentrated under reduced pressure as above to afford: Fraction 1 (20% MeOH, 60 mL, 17 mg), fraction 2 (40% MeOH, 120 mL, 1.01 g), fraction 3 (60% MeOH, 60 mL, 18 mg), fraction 4 (80% MeOH 60 mL, 5.6 mg), fraction 5 (100% MeOH, 60 mL, 5 mg). Fraction 2 contained product as an off-white solid, 97% yield. Unreacted furosemide eluted in fraction 4.
Melting point (Mp) decomposed above 200° C. 1H NMR (DMSO-d6) □ 8.42 (s, 1H), 7.54 (s, 1H), 7.07 (s, 2H), 6.75 (s, 1H), 6.35 (m, 1H), 6.26 (m, 1H), 4.39 (d, J=6.4 Hz, 2H). 13C (DMSO-d6) d 170.8 (C12), 152.0 (C9), 151.8 (C6), 142.2 (C1), 133.8 (C10 or 7), 133.6 (C4), 125.6 (C8), 115.1 (C10 or 7), 112.2 (C2 or 3), 110.3 (C11), 107.2 (C2 or 3), 39.5 (C5). Metals analysis (total metals by ICP) 3.3000% Mg; Theory (Mgfur2.H2O) 3.41% Mg. Total hardness analysis (EDTA titration, Calmagite indicator) 3.3%. Elemental analysis (C, H, N, Cl) 41.10, 3.20, 8.00, 9.72%; Theory (Mgfur2.H2O) 41.08, 3.16, 7.98, 10.10%.
The structure of Bis(furosemide)magnesium is as follows:
Partition Coefficients, Distribution Coefficients and pKa for T3 and Tetracycline Complexes and Reference Drugs
Determination of pKa and log P was done by potentiometry and spectrophotometry. The potentiometric method includes the use of expert software to calculate pKa and log P from simple acid and base titrations of the analytes. The pKa was first determined by weighing approximately 2 mg of pure substance into an assay vial. Ionic strength was adjusted with 0.15M KCl and water was added to dissolve the compound followed by an acid or base titrant to drop or raise the pH to the desired starting value. The solution was then titrated with acid (0.5N HCl) or base (0.5N NaOH) to the final pH. Approximate pKa values were displayed and later refined to exact data.
For (T3)2Zn, which is sparingly soluble in water, the pKa was determined in mixtures of water and DMSO cosolvent. A minimum of three ratios of water/DMSO was titrated to obtain psKa (apparent pKa in the presence of cosolvent). The aqueous PKa was determined by extrapolation using the Yasuda-Shedlovsky technique.
The log P was determined by a titration in the presence of octanol (water saturated). The pKa in water and the apparent pKa in the presence of octanol (poKa) were compared and the log P determined. Ion-pairing (partitioning of a charged species into octanol, termed log P+ or log P−) were determined with an additional titration in the presence of another volume of octanol. Using experimentally determined pKa and log P, a drug lipophilicity profile (log D vs. pH) was calculated. The log D7.4 was determined from this profile at pH 7.4.
The spectrophotometric method used a fiber optics dip probe, a UV light source (pulsed deuterium lamp) and a photodiode array detector to automatically capture the absorption spectra of the sample solution in the course of adding an acid or base solution.
Up to a 10 mM stock solution was prepared by dissolving several 0.5 mg samples in 0.5-1.0 mL of water or cosolvent. An adequate amount of stock is pipetted into the vial for titration. The dip probe is in the assay vial and aqueous 0.15 M KCl solution is added to cover the dip probe. Acid or base is added to bring the pH to the desired starting value. Over the chosen pH range, the spectra changes due to ionization were captured by the photodiode array detector for subsequent analysis. Target Factor Analysis (TFA) was applied to deduce the pKa values of the sample and resolve the major absorptivity spectra of the reducing species. The aqueous pKa was determined by extrapolation using the Yasuda-Shedlovsky technique. The pKa values obtained from spectrophotometric experiments are in excellent agreement with those derived from potentiometric titrations.
Bioavailability Study
Title: Assessment of absorbance and effect of a hormone complex supplementation in Sprague Dawley rats
Test subjects: Fifteen young female Sprague Dawley rats (180-225 gms) were used. These rats were obtained from a commercial source (Harlan Laboratory Animals, Dublin, Va.), housed in the Vivarium at Litton Reeves Hall (Division of Laboratory Animal Resources), in groups of three in polypropylene shoebox cages. Water was available ad libitum. Rats were fed certified rodent chow ad libitum. After arrival, the health of rats was assessed and animals were placed in quarantine for a minimum of five days, during which time, general health was assessed. At the end of quarantine, rats were moved to permanent animal quarters for access and study.
Study design: This is a study to compare the absorption and effect of three hormones administered to rats orally directly into the esophagus. T3-Zinc and T3-Magnesium were synthesized, which are the two test compounds. Triiodothyronine, free acid (T3) was the positive control. The three compounds were formulated, separately, into gelatin capsules, with a total dose of 108±12 μg/kg administered. In order to avoid acid degradation of the test compounds in the rat stomach, metal oxides were added to the formulation—to the T3-magnesium compounds, 1.08±0.13 mg of magnesium oxide was added and to the T3-zinc compounds, 106±13 μg of zinc oxide was added. Another T3 control was included, where 145±50 μg of zinc oxide was added to T3, free acid.
For dosing and blood collection, three rats per replicate were anesthetized. A baseline blood sample was collected, by retroorbital sampling. Then, compound was administered orally by gavage tube. Following administration, blood samples (500 μL) were collected at 0.5, 1, 2, 2.5, 5 hours after dosing. Blood samples were centrifuged, the sera removed and the sera kept on ice and then analyzed for serum triiodothyronine levels.
Analysis of samples: Serum triiodothyronine levels were determined by RIA.
Results: The individual serum T3 levels from each group of rats were averaged and a plot of T3 concentration (ng/mL) vs. hour was produced. The plot is shown in
Large Molecule Example:
Preparation of Interference RNA
Interference RNA was prepared using a modified New England Biolabs Litmus 28i RNAi bidirectional transcription vector. A 922 bp bovine serum albumin cDNA fragment was introduced into the BglII and Stul sites of the Litmus RNAi vector. The target RNAi transcript was produced by in vitro transcription with T7 RNA Polymerase to yield 1 mg/ml. The RNA was then divided into 50 ug samples and freeze dried.
The following examples illustrate the use as starting materials compounds that belong to any of the oligonucleotide, phosphate or phosphonate chemical classes. The drug will usually be available as the sodium, potassium or quaternary ammonium salt. DMSO is a preferred solvent and depending on the drug, gentle heating may be required, followed by the addition of a buffer solution and precipitation from alcohol.
Preparation of RNA:Magnesium Inner Coordination (Covalent) Complex
Approximately 50 μg of iRNA was dissolved in 100 μL of anhydrous DMSO at 50° C. A stock solution of 4 mM magnesium chloride was prepared by dissolving 19 mg of anhydrous magnesium chloride in anhydrous DMSO. Three separate reactions were run where 15 μL, 30 μL and 60 μL of 4 mM magnesium chloride was added to three separate solutions of iRNA in DMSO. The solutions were allowed to set at room temperature with occasional vortex mixing for 90 minutes at which time 20 μL of 7.5 M aqueous ammonium chloride followed by 400 μL of RNAse free ethanol was added and vortex mixed. The product was allowed to precipitate out of solution over 1 hour, centrifuged and the liquid decanted from the pellet. The pellet was washed with 100 μL of RNAse free ethanol, vortex mixed, centrifuged and the ethanol supernatant decanted off the pellet. The resultant colorless pellet was air-dried for several minutes before testing in the isoelectric focusing gel.
Preparation of Magnesium:RNA Outer Coordination (Ionic) Complex
The preparation for the ionic magnesium:RNA complex followed the procedure for the covalent analog exactly except the stock magnesium chloride solution was prepared in RNAse free water instead of anhydrous DMSO. The resultant colorless pellet was air-dried for several minutes before testing in the isoelectric focusing gel.
Preparation of RNA:Zinc Inner Coordination (Covalent) Complex
The preparation for the covalent zinc:RNA complex followed the procedure for the magnesium analog exactly except a stock zinc chloride solution was prepared instead of a stock magnesium chloride solution. The resultant colorless pellet was air-dried for several minutes before testing in the isoelectric focusing gel.
Preparation of Zinc:RNA Outer Coordination (Ionic) Complex
The preparation for the ionic zinc:RNA complex followed the procedure for the covalent analog exactly except the stock zinc chloride solution was prepared in RNAse free water instead of anhydrous DMSO. The resultant colorless pellet was air-dried for several minutes before testing in the isoelectric focusing gel.
Isoelectric Focusing Gel Experiment
This is a novel approach of using IEF gels to monitor for the anticipated modification of the iRNA target.
Interpretation of IEF Experiments
The IEF experiment showed that magnesium RNA complexes prepared in anhydrous (A) conditions with three concentrations of magnesium chloride produced covalent complexes in approximately 50% yield. Magnesium RNA complexes prepared in aqueous (W) conditions with three concentrations of magnesium chloride produced ionic complexes. The zinc RNA complex prepared in anhydrous (A) conditions with zinc chloride produced a covalent complex in approximately 50% yield. The zinc RNA complex prepared in aqueous (W) conditions with zinc chloride produced an ionic complex.
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