Patent Application
20080293796
1. Field of the Invention
The present invention relates generally to the fields of pharmacology and pharmaceutics. More particularly, it concerns pharmaceutical compositions that include a benzimidazole, a polyol, and a dipolar aprotic solvent. It also concerns pharmaceutical compositions that include a benzimidazole, an oil, a dipolar aproptic solvent, and a surfactant.
2. Description of the Related Art
The identification of improved methods of drug delivery is crucial in the treatment of a vast number of human diseases. In particular, even though scientific studies might have identified the potential usefulness of a certain drug in the treatment of a particular condition, treatment options may be limited if proper formulations of the drug to facilitate delivery of the agent to the diseased tissue are not identified.
One disease wherein pharmaceutics has played a significant role is cancer. Cancer is the second leading cause of death in the United States. Half of all men and one-third of all women in the United States will develop cancer during their lifetimes. Today, millions of people are living with cancer or have had cancer. The sooner a cancer is identified and treatment initiated, the greater are the chances for survival.
Pharmaceutical preparations that are effective against cancer comprise an emerging and expanding area of research and potential commercial development. Pharmaceutical agents are being developed that can delay or arrest development of cancer, and the development of precancerous lesions into cancers. Precancerous lesions include, for example, lesions of the breast that can develop into breast cancer, lesions of the skin that can develop into malignant melanoma or basal cell carcinoma, colonic adenomatous polyps that can develop into colon cancer, cervical dysplasia that can develop into cervical cancer, premalignant lesions of the oropharynx that can develop into head and neck cancer.
The search for drugs useful for treating and preventing neoplasias in their earliest stages is intensive because chemotherapy and surgery alone are often ineffective, and current cancer chemotherapy has severe side effects. Such preventive treatment is also potentially useful for recovered cancer patients who retain a risk of cancer recurrence, and even for cancer patients who would benefit from compounds that selectively induce apoptosis in neoplastic, but substantially not in normal cells.
Induction of apoptosis is one mechanism by which pharmaceutical agents can kill cancer cells. Apoptosis, sometimes referred to as “programmed cell death,” naturally occurs in many tissues in the body. It plays a critical role in tissue homeostasis, that is, it ensures that the number of new cells produced are correspondingly offset by an equal number of cells that die. Apoptosis is especially pronounced in self-renewing tissues such as bone marrow, immune cells, gut, and skin.
Standard chemotherapeutics can promote apoptosis not only in cancer cells, but also in normal human tissues. These agents often have particularly severe effect on tissues where apoptosis is especially pronounced (e.g., hair, gut, and skin). Thus, standard chemotherapeutics are inappropriate for cancer prevention, particularly if chronic administration is indicated.
Benzimidazoles (BZs) are a broad-spectrum class of antihelmintics that display excellent activity against parasitic nematodes and, to a lesser extent, against cestodes and trematodes. BZs have also been shown to be very effective antiprotozoal agents that also have antifungal activity. It is currently believed that BZs exert their cytotoxic effects by binding to the microtubule system and disrupting its functions (Lacey, 1988; Friedman and Platzer, 1980). The suggestions that tubulin is a target for BZs has been supported by the results of drug-binding studies using enriched extracts of helminth and mammalian tubulin (Lacey, 1988). Moreover, competitive drug-binding studies using mammalian tubulin have shown that BZs compete for colchicine binding and inhibit growth of L1210 murine leukemia cells in vitro (Friedman and Platzer, 1978; Lacey and Watson, 1989). However, BZs are selectively toxic to nematodes when administered as antihelmintics but are not toxic to the host. In contrast, BZs suppress the in vitro polymerization of mammalian tubulin. Differences in both the affinity between the host and parasite macromolecules for BZ (Russell et al., 1992; Kohler and Bachmann, 1981) and the pharmacokinetics of BZs between the host and the parasite have been suggested as responsible for the selective toxicity of BZs (Gottschall et al., 1990) but the actual molecular basis of this selective toxicity remains unclear.
Mebendazole (MZ), or 5-benzoyl-2-benzimidazole carbamic acid methyl ester, is a member of the BZ class of compounds. Recently, MZ has been found to induce mitotic arrest and apoptosis by depolymerizing tubulin in non-small cell lung cancer cells. (Sasaki et al., 2002). MZ has also been found to elicit a potent antitumor effect on human cancer cell lines both in vitro and in vivo (Mukhopadhyay et al., 2002).
MZ was first introduced for the treatment of roundworm infections as a result of research carried out by Brugmans et al. (1971). It is the prototype of a series of broad-spectrum anthelmintics widely used in both animals and man (Michiels et al., 1982) as broad-spectrum anthelmintics for animal and human use (Van den Bossche et al., 1982). Related BZ derivatives with anthelmintic properties include albendazole and flubendazole.
MZ is highly effective in ascariasis, intestinal capillariasis, enterobiasis, trichuriasis, and hookworm (Ancylostoma duodenale and Necator americanus) infection as single or mixed infections. The drug is active against both larval and adult stages of the nematodes that cause these infections, and it is ovicidal for Ascaris and Trichuris (Keystone and Murdoch, 1979; Van den Bossche et al., 1982). Immobilization and death of susceptible gastrointestinal organisms occurs slowly, and clearance from the gastrointestinal tract may not be complete until a few days after treatment with MZ. Together with albendazole, MZ has shown some promise in the treatment of hydatid disease (Wilson et al., 1987).
MZ causes selective disappearance of cyoplasmic microtubules in the tegumental and intestinal cells of affected worms. Secretory substances accumulate in Golgi areas, secretion of acetylcholinesterase and uptake of glucose are impaired, and glycogen is depleted. These effects of MZ are not noted in host cells. MZ has a high affinity for parasite tubulin in vitro, but it also binds to host tubulin. The biochemical basis for its selective action is thus unclear (see Van den Bossche, 1981; Watts et al., 1982).
The conventional formulation of MZ is a tablet form. Tablets of MZ are poorly and erratically absorbed, and concentrations of the drug in plasma are low and do not reflect the dosage taken (Witassek et al., 1981). This is because MZ is highly lipophilic, with an aqueous solubility of less than 1 .mu.g/ml. As a result, the conventional formulations of MZ result in low bioavailability of the drug and erratic absorption from the gastrointestinal tract. Many other BZs and BZ derivatives are also highly lipophilic and erratically absorbed from the gastrointestinal tract.
Therefore, there is a recognized need for improved oral pharmaceutical compositions of BZs and BZ derivatives, such as MZ, that result in greater bioavailability of the drug, are needed to treat systemic diseases such as cancer and deep-seated parasitic diseases with extraintestinal manifestations. In addition, parenteral formulations that result in greater release of BZs and BZ derivatives compared to conventional formulations would also be beneficial in treating these systemic conditions. Pharmaceutical compositions such as these would result in greater concentration of the drug in the bloodstream, and consequentially greater efficacy and therapeutic benefit. The present invention fulfills this long-standing need and desire in the art.
The present invention is based on the development of novel formulations BZ and BZ derivatives that result in improved drug solubility, improved drug release from the formulation, and improved bioavailability. Higher concentration of BZ or BZ derivative in the blood would result in greater therapeutic effect of this class of agents for use in the treatment of disorders in which treatment with BZs or BZ derivatives would be beneficial. For example, in the case of MZ, these diseases would include hyperproliferative diseases such as cancer, and extraintestinal and deep-seated parasitic disease such as hydatid disease.
The inventors have formulated MZ using co-solvency and microemulsion approaches which have yielded a drug concentration of 1.4-3.5 mg/ml, which is a 1,400-3,500 fold increase in solubility compared to conventional formulations of MZ. The inventors have assessed the formulations of MZ by in vitro USP dissolution, in vivo bioavailability studies in rats, and cell growth inhibition in cell line cultures of numerous types of cancer cells. A substantial improvement in release of the MZ from the formulations was demonstrated compared to an unformulated suspension, and bioavailability was improved 130 fold. An enhanced IC50 of MZ from the formulations for cell growth in lung cancer cell lines was demonstrated compared to control formulations dissolved in dimethylsulfoxide and the formulations exhibited favorable characteristics in preclinical pharmacokinetic and pharmacodynamic studies. Therefore, the new BZ formulations are expected to have enhanced efficacy in the treatment of cancer and other hyperproliferative diseases.
Certain embodiments of the present invention are pharmaceutical compositions that include a BZ, a polyol, and a dipolar aprotic solvent. The definition of pharmaceutical composition is discussed in detail elsewhere in this specification, and encompasses a composition that does not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate.
Any BZ, BZ derivative, or combination of BZs is contemplated for use in the pharmaceutical compositions of the present invention. One of skill in the art would be familiar with this broad class of agents, and would understand that the pharmaceutical compositions of the present invention pertain to use of any member or members of this class of drugs. For example, the BZ included in the present pharmaceutical compositions may be a derivative having the formula:
wherein R3 is selected from the group that includes H, carboxyl (—CO2H), hydroxyl, amino, chloro, difluormethoxy, benzoyl, phenyl-thio, pyridinyl, propyl-thio, diphenyl, methoxy(methoxy-dimethyl, p-yridinyl)methyl-(sulfonyl), fluorophenylmethyl-2-chloro, propenyl, chloroprophyl or esters (—CO2R4) wherein R4 is selected from the group that includes alkoxy, haloalkyl, alkenyl, and cycloalkyl, wherein the alkyl groups have from 1-8 carbons, or CH3CH2(OCH2CH2)n—, or CH3CH2CH2(O CH2CH2CH2)n, or (CH3)2CH(OCH(CH3)CH2)n—, wherein n is from 1-3, wherein R1 is OH, Cl, SH, carbamate or piperidin-4-yl, and R2 is hydrogen, .alpha.-methylvinyl, 3-chloropropyl or piperidin-4-yl, or the pharmaceutically effective organic or inorganic salts thereof, or mixtures thereof.
In certain embodiments of the present invention, the BZ derivative is methyl 5-benzoylbenzimidazole-2-carbamate (mebendazole, MZ). Alternatively, in other embodiments, the BZ derivative may be methyl 5-(phenylthio)-2-benzimidazole carbamate (fenbendazole) or 5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-ben-zimidazole (omeprazole).
In certain other embodiments, the BZ derivative in the pharmaceutical compositions may be
Alternatively, the BZ derivative of the pharmaceutical compositions may be
In still other embodiments, the pharmaceutical composition includes a BZ derivative that is
The pharmaceutical compositions of the present invention contemplate use of any polyol. One of ordinary skill in the art would be familiar with the class of agents known as polyols, and would understand that any of these agents may be included in the pharmaceutical compositions of the present invention. For example, the polyol may be polyethylene glycol (PEG) 200, PEG 300, PEG 400, PEG 600, 1,2-propylene diol, glycerol, or ethylene glycol. In certain embodiments, the polyol may be PEG 400. The pharmaceutical compositions of the present invention can include a single polyol, or more than one polyol.
The pharmaceutical compositions of the present invention include dipolar aprotic solvents. A dipolar aprotic solvent is a solvent with a comparatively high relative dielectric constant, greater than about 15, and a sizable permanent dipole moment, that cannot donate suitably labile hydrogen atoms to form strong hydrogen bonds. The term is something of a misnomer, since such solvents are usually not aprotic but protophilic and, at most, weakly protogenic. One of ordinary skill in the art would be familiar with the class of agents typically known as dipolar aprotic solvents, and would understand that any of these agents may be included in the pharmaceutical compositions of the present invention.
For example, the dipolar aprotic solvents include N,N-dimethylacetamide (DMA) or dimethylsulfoxide (DMSO). In certain embodiments, the pharmaceutical compositions include N,N-dimethylacetamide as the dipolar aprotic solvent. In certain other embodiments, the dipolar aprotic solvent is dimethylsulfoxide. In some embodiments, the pharmaceutical compositions include more than one dipolar aprotic solvent. For example, the pharmaceutical compositions may include N,N-dimethylacetamide and dimethylsulfoxide.
The pharmaceutical compositions of the present invention contemplate any ratio of polyol to dipolar aprotic solvent by weight. However, in certain embodiments of the present invention, the ratio of polyol to dipolar aprotic solvent by weight is 3:1. For example, the pharmaceutical compositions of the present invention may include PEG 400 and N,N-dimethylacetamide in a ratio of 3:1 by weight. Alternatively, certain embodiments of the present invention may include PEG 400 and dimethylsulfoxide in a 3:1 ratio by weight.
In some embodiments of the present pharmaceutical compositions, the ratio of polyol to dipolar aprotic solvent is 7:1 by weight. For example, the pharmaceutical may include PEG 400 and dimethylsulfoxide in a ratio of 7:1 by weight, or PEG 400 and N,N-dimethylacetamide in a ratio of 7:1 by weight.
In certain embodiments of the present pharmaceutical compositions, the composition includes water. Any amount of water is contemplated by the present invention. However, in certain embodiments, the relative ratio of polyol:dipolar aprotic solvent:water is 3:1:1 by weight. For example, some embodiments include a pharmaceutical compositions that may include PEG 400:N,N-dimethylacetamide:water in a 3:1:1 ratio by weight. Alternatively, other embodiments of the present pharmaceutical compositions include PEG 400:dimethylsulfoxide:water in a 3:1:1 ratio by weight.
In other embodiments of the present pharmaceutical compositions, the relative ratio of polyol:dipolar aprotic solvent:water is 7:1:2. For example, the pharmaceutical composition may include PEG 400:dimethylsulfoxide:water in a 7:1:2 ratio by weight.
Additional embodiments of the present invention pertain to pharmaceutical compositions that include (1) a BZ; (2) an oil; (3) a dipolar aprotic solvent; and (4) a surfactant. Members of the class of BZ and BZ derivatives are discussed above, and elsewhere in this specification. As discussed above, one of skill in the art would be familiar with this broad class of agents, and would understand that the pharmaceutical compositions of the present invention pertain to use of any member or combination of members of this class of drugs. For example, as discussed above, the BZ derivative may be MZ.
Any oil is contemplated for use in the present invention. One of ordinary skill in the art would be familiar with the wide variety of oils that are available for inclusion in pharmaceutical compositions. For example, the oil may be super refined corn oil, super refined cottonseed oil, olive oil, super refined peanut oil, super refined soybean oil, Captex 200, Captex 355, Miglyol 812, or Myvacet 9-45. In certain embodiments of the present pharmaceutical compositions, the oil is Captex 200. In certain other embodiments of the present pharmaceutical compositions, the oil is Myglyol 812. The pharmaceutical compositions of the present invention also contemplate compositions containing more than one oil or any combination of oils.
Any surfactant is contemplated for use in the present invention. One of ordinary skill in the art would be familiar with the wide variety of surfactants that are available for inclusion in pharmaceutical compositions of the present invention. For example, in certain embodiments, the pharmaceutical compositions of the present invention include Tween 80 as the surfactant. In other embodiments, the pharmaceutical compositions include Arelacel 80 as the surfactant. The pharmaceutical compositions of the present invention may include a single surfactant, or any combination of surfactants.
Surfactants of any hydrophilic-lipophilic balance (HLB) are contemplated for use in the present invention. The HLB of a surfactant is an empirical quantity, on an arbitrary scale, which is a measure of the polarity of a surfactant or mixture of surfactants. It is a widely known and used term which would be very familiar to one of ordinary skill in the art. In general, a high HLB surfactant is a surfactant with an HLB that is 7 or above 7, and a low HLB surfactant is a surfactant with an HLB below 7. More than one surfactant in the composition may provide for versatility, greater stability, and enhanced drug absorption. For example, some embodiments of the present invention include a combination of Tween 80 and Arelacel 80 as the surfactants.
In certain embodiments of the present pharmaceutical compositions, the surfactants used are only high HLB surfactants. Other embodiments of the present pharmaceutical compositions include only low HLB surfactants. Alternatively, the pharmaceutical compositions of the present invention include a mixture of both high HLB surfactants and low HLB surfactants.
As discussed above, the pharmaceutical compositions of the present invention may further be defined as including water. Any amount of water is contemplated in the present pharmaceutical compositions, as long as any amount of the previously defined components is present.
The pharmaceutical compositions of the present invention may further be defined as a microemulsion. A microemulsion is herein defined as a thermodynamically stable dispersion of one liquid phase into another, stabilized by an interfacial film of surfactant. This dispersion may be either oil-in-water or water-in-oil. Microemulsions are often clear solutions, as the droplet diameter may be approximately 100 nanometers or less. However, solutions that are not clear are encompassed within the definition of microemulsion used herein, and any droplet diameter is contemplated in this definition.
Examples of certain embodiments of the present invention include pharmaceutical compositions that include (1) MZ; (2) 10%-60% by weight of an oil; (3) 2%-30% by weight of a dipolar aprotic solvent; (4) 5%-50% by weight of a first surfactant; and (5) 5%-50% by weight of a second surfactant.
The pharmaceutical compositions of the present invention contemplate any concentration of MZ. For example, in certain embodiments the concentration of MZ is greater than 1.4 mg/ml. For example, the MZ concentration may be about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg.ml, about 2.0 mg/ml, about 2.1 mg/ml, about 2.2 mg/ml, about 2.3 mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2.6 mg/ml, about 2.7 mg/ml, about 2.8 mg/ml, about 2.9 mg/ml, about 3.0 mg/ml, about 3.1 mg/ml, about 3.2 mg/ml, about 3.3 mg/ml, about 3.4 mg/ml, about 3.5 mg/ml, or any range of concentration or increments of concentration derivable therein.
In other embodiments, the concentration of MZ is 0.3-3.0 mg/ml. For example, the concentration of MZ may be about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2.0 mg/ml, about 2.1 mg/ml, about 2.2 mg/ml, about 2.3 mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2.6 mg/ml, about 2.7 mg/ml, about 2.8 mg/ml, about 2.9 mg/ml, or any range of concentration or increment of concentration derivable therein. In other embodiments, the concentration of MZ is 0.5 mg/ml to 2.5 mg/ml or 0.3 mg/ml to 3.0 mg/ml.
In certain specific embodiments, the concentration of MZ is about 1.2 mg/ml. In other embodiments, the concentration of MZ is about 1.5 mg/ml. In still other embodiments, the concentration of MZ is about 1.7 mg/ml. In additional embodiments, the MZ concentration is about 1.8 mg/ml. In other embodiments, the MZ concentration is about 1.85 mg/ml. In other embodiments, the MZ concentration is about 1.9 mg/ml. In further embodiments, the MZ concentration is about 2.0 mg/ml. In still further embodiments, the MZ concentration is about 2.1 mg/ml. In certain other embodiments, the concentration of MZ is about 2.3 mg/ml. In further embodiments, the MZ concentration is about 2.6 mg/ml.
The concentration of BZ in the pharmaceutical compositions of the present invention may be determined by any method known to those of skill in the art. One of ordinary skill in the art would be familiar with the range of techniques used to determine concentration of a BZ or BZ derivative in a composition. For example, the concentration of BZ may be determined by HPLC. In certain embodiments wherein the BZ derivative is MZ, HPLC may be used to determine the concentration of MZ. Use of HPLC to determine concentration of MZ in a pharmaceutical composition of the present invention is discussed further in the specification below.
In some embodiments of the present invention, the pharmaceutical compositions may include water. Any amount of water is contemplated in the present invention, as long as the other required constituents are present. In addition, the pharmaceutical compositions of the present invention contemplate the presence of components in addition to those that have been delineated. A greater discussion of potential additional additives is discussed in the specification below.
Some embodiments of the present pharmaceutical compositions include Tween 80 as the first surfactant and Arelacel 80 as the second surfactant. For example, in some embodiments, the pharmaceutical compositions include 10%-60% by weight of Captex 200, 3%-30% by weight of N,N-dimethylacetamide, 5%-50% by weight of Tween 80, 5%-50% by weight of Arelacel 80, and 2%-20% by weight of water. In other embodiments, the pharmaceutical composition includes 28.9% by weight of Captex 200, 13.7% by weight of N,N-dimethylacetamide, 28.8% by weight of Tween 80, 28.8% by weight of Arelacel 80, and 13% water.
In certain other embodiments, the pharmaceutical composition includes 10%-60% by weight of Captex 200, 2%-20% by weight of dimethylsulfoxide, 10%-40% by weight of Tween 80, 10%-40% by weight of Arelacel 80, and 10%-20% by weight of water. For example, the pharmaceutical composition may include 50.1% by weight of Captex 200, 12.6% by weight of dimethylsulfoxide, 12.6% by weight of Tween 80, 12.6% by weight of Arelacel 80, and 12.1% by weight of water. The pharmaceutical composition may also include 42.6% by weight of Captex 200, 10.7% by weight of dimethylsulfoxide, 16.1% by weight of Tween 80, 16.1% by weight of Arelacel 80, and 14.5% by weight of water. In other examples, the pharmaceutical compositions includes 38.4% by weight of Captex 200, 9.6% by weight of dimethylsulfoxide, 19.2% by weight of Tween 80, 19.2% by weight of Arelacel 80, and 13.6% by weight of water. In further examples, the pharmaceutical composition includes 34.7% by weight of Captex 200, 8.7% by weight of dimethylsulfoxide, 21.7% by weight of Tween 80, 21.7% by weight of Arelacel 80, and 13.2% by weight of water.
The pharmaceutical compositions of the present invention may also include 10%-60% by weight of super refined soybean oil, 2%-20% by weight of dimethylsulfoxide, 10%-40% Tween 80, 10%-40% Arelacel 80, and 0.5%-15% by weight of water. For example, the pharmaceutical composition may include 56.5% by weight of super refined soybean oil, 14.1% by weight of dimethylsulfoxide, 14.1% by weight of Tween 80, 14.1% by weight of Arelacel 80, and 1.2% by weight of water. In other embodiments of the present invention, the pharmaceutical composition includes 44.2% by weight of super refined soybean oil, 11.1% by weight of dimethylsulfoxide, 16.6% by weight of Tween 80, 16.6% by weight of Arelacel 80, and 11.5% by weight of water. In further embodiments, the pharmaceutical composition includes 40.0% by weight of super refined soybean oil, 10.0% by weight of dimethylsulfoxide, 20.0% by weight of Tween 80, 20.0% by weight of Arelacel 80, and 10.0% by weight of water. In additional embodiments, the pharmaceutical composition includes 35.7% by weight of super refined soybean oil, 8.9% by weight of dimethylsulfoxide, 22.3% by weight of Tween 80, 22.3% by weight of Arelacel 80, and 10.8% by weight of water.
Embodiments of the present invention also include pharmaceutical compositions that include 10%-60% by weight of Miglyol 812, 2%-20% by weight of dimethylsulfoxide, 10%-40% by weight of Tween 80, 10%-40% by weight of Arelacel 80, and 5%-20% by weight of water. For example, the pharmaceutical composition may include 50.4% by weight of Miglyol 812, 12.6% by weight of dimethylsulfoxide, 12.7% by weight of Tween 80, 12.7% by weight of Arelacel 80, and 11.6% by weight of water. In other examples, the pharmaceutical composition includes 42.4% by weight of Miglyol 812, 10.6% by weight of dimethylsulfoxide, 15.9% by weight of Tween 80, 15.9% by weight of Arelacel 80, and 15.2% by weight of water. In other examples, the pharmaceutical compositions include 39.2% by weight of Miglyol 812, 9.8% by weight of dimethylsulfoxide, 19.6% by weight of Tween 80, 19.6% by weight of Arelacel 80, and 11.8% by weight of water. The pharmaceutical composition of the present invention may also include 34.7% by weight of Miglyol 812, 8.7% by weight of dimethylsulfoxide, 21.8% by weight of Tween 80, 21.8% by weight of Arelacel 80, and 13.0% by weight of water.
The present pharmaceutical compositions can be formulated by any method known to those of skill in the art. For example, in certain embodiments the pharmaceutical composition is formulated for parenteral administration to a subject. Parenteral administration is defined as administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.
In other embodiments of the present invention, the pharmaceutical composition is further defined as being suitable for administration through the digestive tract or through non-invasive topical and regional routes. For example, the pharmaceutical preparation may be administered orally. In other embodiments, the pharmaceutical preparation is administered intranasally, topically, locally, by inhalation, by continuous infusion, or by localized perfusion by bathing target cells directly, via a catheter, or via lavage.
Embodiments of the present invention also include drug delivery systems comprising a nanosuspension formulation of particles having a benzimidazole derivate for improved release over time and bioavailability to a cell or tissue in a subject. In some embodiments the drug delivery system is defined as comprising 1) the benzimidazole derivative and, in other embodiments, further including two or more surface stabilizers, such as 2) one or more block copolymers, 3) one or more surfactants and, optionally, 4) water. The nanosuspension may comprise up to about 10% w/v each, preferably about 5% w/v, more preferably, about 1% w/v, of block polymer and surfactant. Also, the benzimidazole derivative may comprise the nanosuspension at any concentration of about 200 mg/ml. In addition the particles comprising the nanosuspension may have substantially the same sizes or diameters or may have a combination of sizes or diameters. In a nonlimiting example a range of particles sizes or diameters may be about 5 nm to about 2000 nm, preferably about 100 nm to about 2000 nm.
More particularly, in certain embodiments the present drug delivery systems may have a nanosuspension formulation comprising a block copolymer that is Pluronic F108 and a surfactant that is Tween 80 although many other block polymers and surfactants work as well. Optionally, the formulation may comprise water. In these embodiments the benzimidazole derivative is methyl 5-benzoylbenzimidazole-2-carbamate (mebendazole) at any concentration up to about 200 mg/ml.
In other embodiments of the present invention, the drug delivery systems are defined as being suitable for defining a nanosuspension of a benzimidazole derivative as having maximum therapeutic efficacy for a treatment regimen of a tissue in a subject. Sizes of the particles of the benzimidazole derivative comprising the nanosuspensions are adjusted and/or selected based on pharmacokinetic parameters of the benzimidazole derivative in the tissue to be treated. For example, particle size is adjusted within a range of about 5 nm to about 2000 nm. Thus, the adjusted particle sizes(s) are effective together to maximize the therapeutic efficacy during treatment thereby defining the nanosuspension defined thereby. The pharmacokinetic parameters used to determine the adjustment of the particle size may be one or more of concentration, retention, bioavailability, biodistribution, or toxicity of the benzimidazole derivative in the particular tissue. In a further embodiment the nanosuspension is formulated with the adjusted particle sizes and administered parenterally to the individual during the treatment regimen.
In a related embodiment of the present invention, the drug delivery systems are further defined as being suitable for increasing the bioavailability of a benzimidazole derivative in a tissue for treatment of a pathophysiological condition in a subject. The nanosuspension of particles is formulated such that the particles have a range of sizes or diameters. Retention of the benzimidazole derivative within the particles increases as the particle size or diameter increases. Upon administering the nanosuspension to the subject, release of the benzimidazole derivative from the particles into the tissue is continuous and sustained over a range of time as determined by the particle sizes. For example the nanosuspension may be administered parenterally. In particular embodiments the particle sizes or diameters may range from about 5 nm to about 2000 nm. Thus, it is contemplated that the drug delivery systems of the present invention are suitable to treat a pathophysiological condition such as cancer, for example, a lung cancer or liver cancer.
In another related embodiment, the drug delivery systems are further defined as being suitable for treating a cancer a subject in need thereof. Upon administration of the nanosuspension to the subject, release of the benzimidazole derivative from particles comprising the nanosuspension into a cancerous tissue provides a therapeutic effect thereby treating the same. In a further particular embodiment, prior to administration, the particles comprising the nanosuspension are formulated with a range of sizes or diameters such that retention of the benzimidazole derivative within the particles increases as the particle diameter increases, thereby increasing the bioavailability thereof to the tissue. Particularly, a nanosuspension may comprise particles with diameters ranging from about 5 nm to about 2000 nm which may be administered parenterally.
Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
As used herein, the term “a” or “an”, when used in conjunction with the term “comprising” in the claims and/or the specification, may refer to “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
As used herein, the term “or” in the claims refers to “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used herein, the terms “pharmaceutical,” “pharmaceutically,” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate. The term “pharmaceutical” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-cancer agents or anthelmintics, can also be incorporated into the compositions.
As used herein, the term “nanosuspension” refers colloidal dispersions of nanometer-sized drug particles stabilized by surface modifying agents. They are promising drug delivery systems for poorly water-soluble drugs as well as poorly oil-soluble drugs. One great advantage of nanosuspensions over other nanoformulations such as liposomes, micells, microemulsions, etc. is that nanosuspensions only contain pure drug without carriers or organic solvents, which largely reduces potential toxic risk of the formulation. In addition, a high concentration of drug can be prepared using nanosuspensions for dosing. Therefore, less administration volume will be needed. Nanosuspensions are suitable for a variety of drug delivery systems such as oral, parenteral, ocular, pulmonary and targeted drug delivery.
As used herein, the term “subject” refers to any recipient of a benzimidazole derivative via the pharmaceutical compositions or drug delivery systems described herein.
The present invention seeks to exploit the inventors' discovery by providing pharmaceutical compositions of BZs and BZ derivatives that have improved and predictable bioavailabilities. BZs and BZ derivatives often have limited and erratic oral bioavailability due to poor solubility of the compound in aqueous medium. As a result, these drugs cannot be efficiently delivered in convention dosage forms. For example, MZ has a solubility in aqueous medium of less that 1 μg/ml. The inventors have discovered certain oral and parenteral formulations of BZs and BZ derivatives, such as MZ, using co-solvency and microemulsion approaches, respectively. The parenteral formulation of MZ was developed to circumvent the drawback of extensive first-pass metabolism of MZ known to be associated with oral administration. These formulations can be applied for the therapy of diseases wherein BZs and BZ derivatives formulated for increased bioavailability may be efficacious, such as hyperproliferative diseases, neoplastic disease and extraintestinal parasitic diseases.
Benzimidazoles (BZs) are broad-spectrum antihelmintics that display excellent activity against parasitic nematodes and, to a lesser extent, against cestodes and trematodes. BZs have also been shown to be very effective antiprotozoal agents and to have antifungal activity. It is currently believed that BZs exert their cytotoxic effects by binding to the microtubule system and disrupting its functions (Lacey, 1988; Friedman and Platzer, 1980). The suggestion that tubulin is a target for BZs has been supported by the results of drug-binding studies using enriched extracts of helminth and mammalian tubulin (Lacey, 1988). Moreover, competitive drug-binding studies using mammalian tubulin have shown that BZs compete for colchicine binding and inhibit growth of L1210 murine leukemia cells in vitro (Friedman and Platzer, 1978; Lacey and Watson, 1989). However, BZs are selectively toxic to nematodes when administered as antihelmintics but are not toxic to the host. In contrast, BZs suppress the in vitro polymerization of mammalian tubulin. Differences in both the affinity between the host and parasite macromolecules for BZ (Russell et al., 1992; Kohler and Bachmann, 1981) and the pharmacokinetics of BZs between the host and the parasite have been suggested as responsible for the selective toxicity of BZs (Gottschall et al., 1990) but the actual molecular basis of this selective toxicity remains unclear.
Benzimidazoles and derivatives of benzimidizoles are defined herein to have the formula:
wherein R is selected from the group consisting of H, carboxyl (—CO2H), hydroxyl, amino or esters (—CO2R′) wherein R′ is selected from the group consisting of alkoxy, haloalkyl, alkenyl, and cycloalkyl wherein the alkyl groups have 1-8 carbons or CH3CH2(OCH2CH2)n— or CH3CH2CH2(OCH2CH2CH2)n or (CH3)2CH(OCH(CH3)CH2)n where n is from 1-3 and the pharmaceutically acceptable organic or inorganic addition salts thereof; wherein R3 is selected from the group consisting of H, carboxyl (—CO2H), hydroxyl, amino, chloro, difluormethoxy, benzoyl, phenyl-thio, pyridinyl, propyl-thio, diphenyl, methoxy(methoxy-dimethyl, pyridinyl)methyl-(sulfonyl), fluorophenyl methyl-2-chloro, propenyl, chloroprophyl or esters (—CO2R4) wherein R4 is selected from the group consisting of alkoxy, haloalkyl, alkenyl, and cycloalkyl, wherein the alkyl groups have from 1-8 carbons, or CH3CH2(OCH2CH2)n— or CH3CH2CH2(OCH2CH2CH2)n— or (CH3)2CH(OCH(CH3)CH2)n—, wherein n is from 1-3, wherein R1 is OH, Cl, SH, carbamate or piperidin-4-yl, and R2 is hydrogen, .alpha.-methylvinyl, 3-chloropropyl or piperidin-4-yl, or the pharmaceutically effective organic or inorganic salts thereof, or mixtures thereof. For R4, the preferred alkyl groups are straight chain, the preferred the halogen is substituted on the terminal carbon, the preferred halogen is chlorine, the preferred cycloalkyl groups are those having 3-6 carbon atoms, and the cycloalkyl groups also include those which are substituted on an alkyl chain, 2-cyclopropylethyl, cyclopropylmethyl, 2-cyclopropylpropyl or 2-cyclopropylpropyl or cyclohexylmethyl.
BZs have some anti-tumor growth properties in vitro (WO 98/513304; WO 98/32440). The efficacy of BZs in vivo as an anti-tumor treatment has been limited to tumors already in regression following chemotherapeutic treatment.
Alternative benzimidazoles are: fenbendazole, albendazole, albendazole sulfone, oxibendazole, rycobendazole, thiabendazole, oxfendazole, flubendazole and carbendazim. Alternative anti-helminthic drugs are the imidazoles: niridazole and levimasole, the piperazines: piperazine and diethylcarbamazine, the isothiocyanates: amoscanate and CGP 6140. In addition, suramin, ivermectin, hycanthone, metrifonate, oxamniquine and praziquantel are anti-helminthic drugs.
There is evidence, as discussed above, that BZs or BZ derivatives may be useful in the treatment of hyperproliferative diseases, such as cancer. In particular, the use of BZs in the treatment of cancer is discussed in detail in U.S. patent application Ser. No. 10/043,877, which is specifically incorporated by reference herein. In addition, MZ has also been found to elicit a potent antitumor effect on human cancer cell lines both in vitro and in vivo (Mukhopadhyay et al., 2002; Liang et al., 2002). Mukhopadhyay et al., 2002, and Liang et al., 2002, are specifically incorporated by reference herein.
Of particular interest are patients that have wild-type tumor suppressor (e.g., p53) function. The tumor suppressor status of the tumor cells can be determined using any conventional methods, examples of which are described below. Patients may, but need not, have received previous chemo-, radio-, or gene therapies. Optimally, patients will have adequate bone marrow function (defined as peripheral absolute granulocyte count of >2000/mm3 and platelet count of 100,000/mm3), adequate liver function (bilirubin ≦1.5 mg/dl) and adequate renal function (creatinine <1.5 mg/dl).
Patients with cancer may be treated with a pharmaceutically acceptable form of BZ or a functional analog thereof. This administration could be in the form of, for example, an intratumoral injection, or indeed, any other method of application that is routinely used and well known to one of skill in the art, e.g., oral, systemic, local, regional. A biopsy of the lesions to be injected may be performed and the tissue stored for immunohistochemistry analyses.
The dose of BZ typically will be reconstituted into a pharmaceutically acceptable form immediately prior to administration. The dose of BZ will be determined depending on the clinical condition to be treated. For example, if the disease is cancer, the starting dose will be approximately 0.1 to 1 mg BZ/kg body weight. Of course, this may vary depending on the disease to be treated, the size of the tumor, the rate at which the tumor is growing, etc.
The objective of the present invention was to develop both parenteral and oral formulations of BZ and BZ derivatives, such as MZ, in order to achieve improved and predictable bioavailabilities. The invention stems from the desire to overcome the problem of limited and erratic oral bioavailability due to poor solubility in aqueous medium of many BZs and BZ derivatives. The parenteral and oral formulations of BZs described in greater detail below were developed using co-solvency and self-emulsifying/microemulsion approaches, respectively. The parenteral formulation of BZ were developed to circumvent the drawback of the extensive first-pass metabolism of MZ and other BZs known to be associated with oral administration.
The inventors have investigated the solubility of MZ in various solvents, such as N,N-dimethylacetamide (DMA), DMSO, and polyethylene glycol-400 (PEG), as discussed in greater detail in the examples below. As primary solvents, the solvents would be miscible in secondary solvents, examples of which are normal saline, dextrose in water (5% or 10%), and water. These solvents are examples of vehicles in which BZs such as MZ could be suitably solubilized, yet be safe for human administration, alone or in combinations with other drugs. The solubility of BZ in individual solvent vehicles is shown in Table I below.
Virtually no MZ is absorbed through the intestinal tract after oral administration, making it impossible to even investigate its use as an oral antimicrobial against systemic infections. Parenteral administration would therefore be the logical approach to evaluate MZ and other BZs as therapy for deep-seated, systemic fungal infections and also in the treatment of cancer and other hyperproliferative diseases.
Pharmaceutical compositions of the present invention will include an effective amount of a BZ or a BZ derivative or a mixture thereof that is clinically determined to be useful in the treatment of the particular disease under consideration. For example, an effective amount of a BZ or a BZ derivative in a patient with cancer may be an amount that promotes expression of wild-type tumor suppressor genes, and/or inhibits angiogenesis. Alternatively, an effective amount of a BZ or BZ derivative or mixture thereof may be an amount that promotes death of parasites and/or regression of clinical findings associated with the infection. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier.
In cancer patients, the access to parenteral BZs such as MZ will be particularly important, since their intestinal absorption is often perturbed after chemotherapy, aggravating the already erratic intestinal absorption of various medications. The parenteral route will also make it possible to circumvent unpredictable first-pass metabolic effects in the liver, well known to alter the bioavailability of numerous pharmacologically active agents after oral dosing. Further, the availability of MZ for effective and reliable systemic administration will for the first time make it possible to clinically compare the activity of MZ against other treatments for cancer and deep-seated fungal infections.
In addition to the compounds formulated for parenteral administration, such as those for intravenous, subcutaneous, or intramuscular injection, other pharmaceutically acceptable forms include, solutions suitable for oral administration. The term “oral administration” includes any form of administration of drug through the oral cavity and into the gastrointestinal tract. The pharmaceutical compositions of the present invention may be formulated for delivery to a subject by any means. For example, the compositions may be formulated as mouthwashes, mouthrinses, solutions for administration through a nasogastric tube, and the like.
As discussed in the summary of the invention, the active compounds of the present invention will often be formulated for parenteral administration, e.g., formulated for injection by any route other than through the digestive tract, such as via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. The definition of parenteral administration is discussed further in the summary of the invention. Typically, the compositions can be prepared as injectables, either as liquid solutions or suspensions for any parenteral but intravenous routes; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitable for mixture with a surfactant, such as hydroxypropylcellulose or polysorbate. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions, and formulations including sesame oil, peanut oil or aqueous propylene glycol or other solvent as discussed in the examples below. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be chemically and physically stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
The active compounds may be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Preparation of cosolvency formulations and microemulsion formulations of BZs or BZ derivatives is discussed in greater detail in the examples below.
The proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
In certain cases, the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in gels, creams, and lotions. These forms may be used for treating skin-associated diseases, such as various sarcomas.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.
For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary. These particular solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
6. Formulation of BZs and BZ Derivatives that are Sparingly Soluble in Water: Co-Solvency Systems
Many BZs are only sparingly soluble in aqueous solutions. As discussed above, it is therefore an aspect of the current invention that the BZ compound be in a formulation which allows for an increased solubilization of the BZ or for a more effective dispersion.
Generation of some of the pharmaceutical compositions of the present invention are based on the principle of cosolvency (Spiegel and Noseworthy, 1963; Yalkowsky and Roseman, 1981). The examples show that pharmaceutical compositions of MZ can be generated without destroying its anti-cancer properties. Further, the animal studies provide evidence that the preferred vehicles are nontoxic and safe for administration and should be acceptable for human administration in the proposed concentrations and total doses to be utilized; indeed, DMA, DMSO, and PG have been used for solubilization of various pharmacologically active agents used in man (NIH Pub. 84-2141, 1984; Weiss et al., 1962; Kim, 1988). The parenteral administration of PEG has been studied in detail in a simian model (Lockard et al., 1979), and PEG has subsequently been used clinically as a (covalently bound) carrier of L-asparaginase in the treatment of lymphocytic leukemia and lymphoma (Keating et al., 1993). DMSO is also extensively used as a cryoprotective agent for low-temperature storage of human bone marrow and peripheral blood derived hematopoietic stem cell preparations to be used for transplantation after high-dose chemotherapy (McGann, 1978; Gorin, 1986; Davis and Rowley, 1990; Gorin, 1992). No serious adverse effects have been experienced from the use of these vehicles. The clinical use of normal saline, dextrose in water (5-70%), and aqueous lipid emulsion are well established means to alter the fluid and electrolyte balance and to supply parenteral nutrition. Normal saline and dextrose in water are extensively used to dilute various medications for parenteral use.
7. Formulation of BZs and BZ Derivatives that are Sparingly Soluble in Water: Microemulsion Systems
The preparation and use of microemulsions in the formulation of drugs, proteins, and the like are known in the art. A microemulsion is herein defined as a thermodynamically stable dispersion of one liquid phase into another, stabilized by an interfacial film of surfactant. This dispersion may be either oil-in-water or water-in-oil. Microemulsions are often clear solutions, as the droplet diameter may be approximately 100 nanometers or less. However, solutions that are not clear are encompassed within the definition of microemulsion used herein, and any droplet diameter is contemplated in this definition.
Surfactants can be added to the aqueous solution to increase solubility and stability of a solution or dispersion. Surfactants, discussed in greater detail in the summary of the invention, are organic molecules which contain both hydrophilic and hydrophobic ends. The hydrophilic end, which is either polar or ionic, dissolves readily in water. The hydrophobic, or non-polar, end, however, does not dissolve in water and will move as far away from water as possible. When a small concentration of surfactant is added to water, the hydrophobic end will immediately rise to the surface or orient towards a less polar group. The addition of surfactants in a BZ solution increase the solubility and/or stability of the BZ-surfactant solution relative to the BZ alone.
Following preparation of the pharmaceutical compositions of the present invention, it may be desirable to quantify the amount of BZ or BZ derivative in the pharmaceutical composition. Methods of measuring concentration of a drug in a composition include numerous techniques that are well-known to those of skill in the art. Selected examples include chromatographic techniques. There are many kinds of chromatography which may be used in the present invention: drug-specific assays, adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer chromatography, gas chromatography, and high performance liquid chromatography (HPLC). One of ordinary skill in the art would be familiar with these and other related techniques.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Mebendazole (MZ, Lot 90K1145), polyethylene glycol (PEG) 400 (Lot 121H0052), N,N-demethylacetamide (DMA) (Lot 129H3498), and dimethylsulfoxide (DMSO) (Lot 94H0358) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Propylene glycol (Lot 940629) and olive oil (Lot CAS8001-25-0) were obtained from Wood Scientific, Inc (Houston, Tex.). Triacetin was purchased from Lancaster Synthesis, Inc. (Windham, N.H.). Super refined cottonseed oil (Lot GAB-217NP), super refined peanut oil (Lot BSS-429NP) and super refined soybean oil (Lot SB4-393NP) were supplied by Croda (Mill Hall, Pa.). Captex 200 (C8/C10 diesters of propylene glycol, Lot 10505-6) and Captex 355 (C8/C10 triglycerides from coconut oil) were provided by Abitec Corporation (Janesville, Wis.). Miglyol 812 (C8/C10 triglycerides from coconut oil, Lot 1995050866) and Myvacet 9-45 (distilled acetylated monoglycerides; Lot 960225) were obtained from Eastman Chemical Products. Tween 80 (polyoxytheylene [20] sorbitan monooleate; Lot 32332) and Arelacel 80 (sorbitan monooleate NF, NLB=4.3, Lot 27255) were supplied by ICI Americans Inc. (Wilmington, Del.). Cremophor RH 40 (PEG40 hydrogenated castor oil, HLB=13.5) was from BASF (Parsippany, N.J.).
An HPLC assay was developed to quantify MZ in the formulations and in the release samples. The assay used a reversed-phase .mu.Bondapak C18 column (300×3.9 mm, 10 μm, Waters Corporation, Milford, Mass.) preceded by a C.sub.18 guard column. The mobile phase, which was filtered and degassed prior to use, consisted of acetonitrile/0.05 M potassium monobasic phosphate, pH 6.5 (40:60, v/v). Prednisone was used as the internal standard (IS). The flow rate was 1.2 ml/min. The solvent delivery system was a ConstaMetric 3500 LDC Analytical Model system. MZ and IS were detected with a SpectroMonitor 3200 programmable multiwavelength UV detector set at 254 nm. Peak area ratios measured by a Milton Ray CI-4100 data module integrator (LDC Analytical) were used to construct standard curves and to determine the concentrations of MZ in the samples. The assay was later modified for the quantification of MZ in blood samples, as described below.
The solubility of MZ in aqueous and in fourteen other solvent media, including six pharmaceutical solvents (methanol, PEG 400, propylene glycol, triacetin, DMA, and DMSO) and eight naturally occurring and synthetic oils (super refined cottonseed oil, olive oil, super refined peanut oil, super refined soybean oil, Captex 200, Captex 355, Myglyol 812, and Myvacet 9-45), were evaluated. The solubility of MZ in each solvent medium was determined by adding excessive amount of MZ to the medium, followed by shaking at room temperature for at least 48 hours. After the equilibrium was reached, an aliquot was withdrawn and filtered through a membrane with a pore size of 0.45 μm. The clear filtrate was diluted with an appropriate volume of DMA and analyzed by the developed HPLC assay.
One hundred milligrams of MZ were dissolved in 11 ml of DMA by warming in a 60.degree. water bath. The solution was then mixed with PEG 400 at ratios of 1:1 to 1:7 (v/v). A final portion of water was added to obtain a final co-solvent formulation with volume ratios of DMA:PEG 400:water at 2:2:1, 1:3:1, and 1:7:2. Using this approach, ten co-solvent systems of various compositions and ratios were evaluated and optimized to achieve the maximal drug solubility for parenteral delivery. The MZ solubility in individual co-solvent formulations was determined by the HPLC assay. The optimal formulation was selected to be administered intravenously (I.V.) as the most bioavailable formulation reference in the preclinical bioavailability study of the selected lead oral microemulsion formulation.
Six self-emulsifying drug delivery systems (SEDDS) were prepared by mixing an oil, a low HLB surfactant, and a high HLB surfactant. In particular, 100 mg of MZ were dissolved in 5.5 ml of DMSO or DMA at 60° C. in a water bath. The solution was then mixed with Tween 80, Aralacel 80, and various types of oils at different ratios.
A clear, transparent formulation was indicative of the formation of a stable microemulsion. Phase diagrams were constructed with six different ratios of the ingredients for each system, by adding a small increment of an aqueous phase (deionized water) to the SEDDS until the system became turbid, reflecting a phase separation, to define the boundary of the composition region for stable microemulsions.
Once the region of stable microemulsions was identified on the phase diagram, microemulsions were prepared by mixing appropriate quantities of the four ingredients (oil, dipolar aprotic solvent, surfactant, and water) with a gentle hand shaking or stirring to ensure a through mixing. MZ, either dissolved in DMA or DMSO, was incorporated by mixing with the other ingredients.
Two optimal microemulsion formulations were selected for further assessment of in vitro drug release. One of the two optimal formulations was evaluated for cell growth inhibition in cell culture models, and for in vivo preclinical bioavailability in rats. The lead microemulsion formulation will be suitable for future evaluation of anti-neoplastic efficacy in mice as well.
The drug release characteristics of the two selected SEDDS formulation were comparatively evaluated with MZ powder and MZ solution in PEG 400. All four formulations were filled manually with a syringe into a hydrophilic airfill soft gelatin capsule (R. P. Scherer, Basking Ridge, N.J.) and the resulting hole was sealed thermally. USP XXII, Dissolution Apparatus 2 (VanKel Industrial, Inc.) was employed to characterize the release kinetics of MZ in vitro. Soft gelatin capsules containing the SEDDS, MZ solution, or MZ powder were individually placed in copper coils to keep the capsules at the bottom of their respective dissolution vessels. The vessels were filled with 500 mL of deionized water containing 0.5% Cremphor RH 40 (CMC=0.039%) at 37.degree. C. Cremphor RH 40 was used to maintain the sink condition of the releasing medium. The release medium was constantly stirred at 50 rpm by a Teflon-coated dissolution paddle. Serial release samples were withdrawn and filtered through a 0.45 μm Millipore filter and analyzed by the HPLC assay.
Studies of Cell Growth Inhibition in nsclc and Skin Cancer Cells Using Formulation E4 and Placebo
Cells were plated in 96-well plates at 5×106 cells/ml of Dulbecco's modified Eagle's medium (DMEM) containing 100 U/ml penicillin, 100 .mu.g/ml streptomycin, and 10% fetal serum albumin, and incubated at 37° C. in a 5% CO2/95% air-humidified incubator for 24 hrs. Cells were incubated for 48 hours after a medium change with fresh DMEM containing serial dilutions of E4 formulation or placebo. Cell survival was monitored with MTT assay. Anti-tumor effects of MZ were also evaluated using cell viability assays (trypan blue counting).
Male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, Id.), weighing 300-400 g, were used after a 7-day acclimation period. Animals were housed under a 12 h light/dark cycle. A permanent catheter was implanted in the right jugular vein of each rat on the day before the study, under the anesthesia with ketamine:acetopromazine:xylazine (50:3.3:3.3 mg/kg). Animals were fasted overnight, while water was allowed ad libitum. On the morning of the experiment, rats were placed in individual metabolism cages.
The rats were randomized into three groups of three or four each. Rats in Group 1 were injected intravenously through the jugular catheter with a single dose (3.25 mg/kg) of the P7 formulation in a cosolvent system. Group 2 received oral microemulsion formulation E4; at a single dose of 5 mg/kg by oral gavage using an animal feeding needle (18×3″W/2-1/4 mm Ball). Group 3 were dosed with MZ aqueous unformulated suspension at 50 mg/kg similarly by oral gavage. The MZ suspension was prepared at 10 mg/ml with a trace amount of glycerin as a wetting agent.
Serial blood samples approximately 0.6 ml each were collected in heparinized tube through the catheter at determined time intervals for up to 24 hours. The blood samples were immediately centrifuged at 14,000 rpm for 3 min and the supernatant, plasma, was transferred into a 1.7-ml microcentrifuge tube and immediately stored at −80° C. until the HPLC assay.
MZ concentrations in plasma samples were determined by the HPLC method described in Example 1 with the following modifications. Briefly, 300 μl of plasma were mixed with 300 μl of acetonitrile to precipitate proteins. The acetonitrile contained 2 μg/mL quinidine (QD) as internal standard. Prednisone was not used as IS for plasma samples, because it was interfered by the broad solvent front from plasma blank. The mixture of plasma and acetonitrile was vortexed and centrifuged, then 100 μl of the supernatant were withdrawn and injected directly onto the HPLC system. The mobile phase consisted of 35% (v/v) acetonitrile in 0.05 M aqueous solution of KH.sub.2PO.sub.4, pH 6.5. The flow rate was 1.2 ml/min, and the effluent was monitored at 313 nm (0.01 AUFS).
Pharmacokinetic parameters were determined by fitting the plasma concentration-time data for each rat by a WinNonlin program. The maximum (peak) concentrations and the time to achieve maximum concentrations (peak time) in plasma were determined from the computer fitted plot. Areas under the concentration vs. time profiles from 0 to the last time point (720 min) of the measured concentration (AUC0→720) was determined by the linear trapezoidal rule, and the AUC from the time of the last measured concentration to infinity (AUC720→infin.) was determined by dividing the last determined concentration by the terminal phase elimination rate constant, which was initially estimated from the least squares slope of the terminal linear segment of the plot. The terminal phase half-life was calculated as 0.693 divided by the terminal phase elimination rate constant. Other non-compartmental pharmacokinetic parameters were generated by area/moment analysis. Total plasma clearance (CLT) was calculated from Dose/AUC, mean residence time (MRT) was calculated from AUMC/AUC, and steady-state volume of distribution (V.sub.ss) was calculated from [(Dose AUMC)/AUC2]. For oral administration, bioavailability (F) was determined by the following equation:
F=[AUC
oral
/AUC
iv]×[Doseiv/Doseoral]×100%
Differences between any two mean values were statistically evaluated using paired or unpaired Student's t-test subsequent to statistical verification that the associated variances were homogeneous or equal.
The retention times of MZ and the IS (prednisone) were 9.3 min and 5.6 min, respectively, under the HPLC eluting conditions aforementioned in Example 1. In particular, the stationary phase was a C18 column, 300×3.9 mm (Waters Corp.), the mobile phase was acetonitrile/0.05 M KH2PO4 (40:60 V.V; pH=6.5), the flow rate was 1.2 ml/min, the detection was UV at 254 nm, for integration a Milton Ray CI-4100 integrator was used. An authentic HPLC chromatogram of MZ is shown in
For the assay modified for plasma samples, the retention time for MZ and the IS (QD) were 10 and 7 min, respectively. On each day of plasma sample assay, a calibration curve was constructed to calculate the MZ concentrations in plasma samples. The linearity of the curve was established in a MZ concentration range of 0.05 to 2.5 μg/ml, with r2≧0.998. The calibration curve was plotted with peak area ratio of MZ/QD versus the known MZ concentrations spiked in rat plasma blanks.
The solubility of MZ in water and in the fourteen tested solvents at 25° C. are compiled in Table I.
MZ is poorly soluble in water with the solubility of 0.025 μg/mL. The MZ solubility in other solvent media, except super refined cottonseed oil, super refined peanut oil, and Captex 355, were all significantly higher than its aqueous solubility, with enhancement factors ranging from 232 to 200,000 in the reference to MZ aqueous solubility.
The MZ was favorably soluble in the six pharmaceutical solvents (methanol, PEG 400, propylene glycol, triacetin, DMA, and DMSO) with enhancement factors of 1,392 to 200,000. PEG 400, DMA, and DMSO yielded the highest three solubilities among the six tested solvents. PEG 400 alone yielded a solubility of 0.528 mg/ml, still much lower than the target of 1.5 mg/mL.
DMA and DMSO freely dissolved MZ, but were not pharmaceutically acceptable to use as straight solvents. The LD.sub.50 of these solvents in rodents are 3.1 gm/kg. intravenously and 7.92 gm/kg orally. The oral and parenteral pharmaceutical products so far approved by FDA contain no more than 40% of DMA or DMSO. Therefore, PEG 400, DMA, and DMSO were selected to formulate MZ parenteral co-solvent systems with various compositions and ratios.
The MZ solubilities in the remaining five oils (olive oil, super refined soybean oil, Captex 200, Miglyol 812, and Myvacet 9-45) were significantly higher than its aqueous solubility, with enhancement factors ranging from 232 to 1,624 (Table I). However, none of them alone could reach the target concentration of 1.5 mg/ml. All the five oils could be used to formulate MZ microemulsions.
Ten co-solvent systems, coded as P1-P10, containing PEG 400 and DMA or PEG 400 and DMSO with various ratios, were systematically evaluated for their capacity to dissolve MZ. The MZ solubility in individual co-solvent systems are tabulated in Table II.
The developed parenteral formulations yielded MZ solubility in the range of 0.46-3.49 mg/ml, 18,360-139,520 times higher than its aqueous solubility. All the formulations except P2 yielded solubilities higher than that in straight PEG 400, in the range of 1.09-6.61 times the MZ solubility in PEG 400. Six among the ten formulations, P3, P4, P7, P8, P9 and P10, yielded MZ solubility higher than 1.44 mg/ml, and were considered promising and suitable for further in vitro and in vivo evaluations. Formulation P7 with MZ solubility of 1.83 mg/ml was selected for future in vivo bioavailability study, based on its lowest content of DMSO and thus the maximal safety among the six promising systems.
Thirty-six microemulsions (A1-A6, B1-B-6, C1-C-6, D1-D6, E1-E6, and F1-F6) were formulated and their capacities to incorporate water and to dissolve MZ were evaluated. These systems contained an oil (super refined soybean oil, Captex 200, Miglyol 812, or Myvacet 9-45), a solvent (DMA or DMSO), a blend of a high HLB Surfactant Tween 80 and a low HLB Surfactant Arelacel 80 (in a ratio of 1:1 or 2:1), and an aqueous phase. Their compositions are given in Tables III-A-III-F. In these Tables, the following codes are used: J=super refined soybean oil; K=Captex 200; M=Miglyol 812; N=Myvacet 9-45; D=DMA; E=DMSO; O=Tween 80; P=Arelacel 80.
Among the formulated microemulsions, twenty-two formulations (A1-A3, B1-B3, C1-C4, D1-D4, E1-E4, and F1-F4) yielded MZ concentrations in the range of 1.5-2.64 mg/ml, meeting the target concentration of 1.5 mg/ml. When the maximum water that could be incorporated was taken into consideration, sixteen of the twenty-two microemulsion formulations, A3, B3, C1-C4, D3-D4, E1-E4, and F1-F4, that incorporated 10% (by weight) or higher of aqueous medium, were considered more stable than the rest formulations. Twelve microemulsions, C1-C4, E1-E4 and F1-F4 were the most promising formulations. Microemulsion E4 was selected for in vitro drug release and future in vivo studies of bioavailability and anti-neoplastic activity. In release study, formulation A4 was also comparatively evaluated. A representative pseudo-ternary phase diagram indicating the compositions of microemulsions E1-E6 is shown in
MZ Release from Microemulsions A4 and E4
The comparative in vitro release kinetics of MZ from drug powder, solution in PEG 400, microemulsion A4 (containing DMA), and microemulsion E4 (the selected oral formulation, containing DMSO), respectively, were evaluated and summarized in Table IV. The cumulative release profiles are presented in
Eleven rats were used for the preclinical bioavailability evaluations of the lead oral microemulsion formulation in three independent runs. The rats were randomly grouped into three treatment groups: (a) I.V. dose of 3.25 mg/kg, n=3, (b) oral E4 microemulsion dose of 4.89 mg/kg, n=4, and (c) oral suspension dose of 50 mg/kg, n=4 (Table V). Table VI shows the pharmacokinetic parameters of MZ from P7, E4 and oral suspension formulations in rats.
The plasma-time profiles of MZ from I.V., E4 and unformulated suspensions were constructed. (
The plasma profiles were analyzed by non-compartmental model with WinNonlin program to derive pharmacokinetic parameters for comparison. The MZ peak concentration from E4 was 856.+−0.155 ng/ml, 6.5 folds of that from suspension, 131.+−0.47 ng/ml. (Table V). The peak time from both dosing were comparable, 210.+−0.60 and 315.+−0.57 min, for E4 and suspension, respectively. The absorption half-lives were similar, 100.+−0.57 and 184.+−0.63 min, for E4 and suspension, respectively. The biological half-life was not statistically different from the different formulations (I.V. solution, E4 microemulsion and suspension) nor by the various routes of administration (I.V. and oral.), ranging from 100-162 min.
The systemic exposure of MZ after the different dosage forms were measured by AUC0-720. When the values were normalized by the respective doses given, the absolute bioavailability of E4 and suspension in reference to I.V. dosing were 30.6% and 0.31%, respectively (Table V). The relative bioavailability of E4 in reference to the unformulated suspension was 98.7. It means that by formulating MZ into the microemulsion formulation, the bioequivalent dose of E4 was about 1/100 of that of the unformulated suspension.
The E4 formulation and placebo were used in studies of cell growth inhibition in skin cancer cell lines (SRB1, A375 and 2237) and non-small cell lung cancer (NSCLC) cell lines. The dose-response curves for cell growth inhibition were established for the range of 1.7-170 ng/ml for skin cancer cells (
Five ml of Mbz nanosuspensions at 100 mg/mL were prepared as follows. Added into a 20 mL scintillation vial were 12.0 g of milling media, 0.47 g of mebendazole, 0.47 mL of 10% Pluronic F108 solution, 0.47 mL of 10% Tween 80 solution, and 3.37 mL of water for injection. The stirring bar was placed into the vial which is in the holder and 3-5 mm above the center of the stir plate. The beads were present for the purpose of reducing particle sizes. The diameters of the employed beads included 0.25-0.5 mm, 0.5-0.75 mm and 1-1.3 mm.
The mixture is stirred at 1,600 rpm for 1) 300 minutes to prepare a nanosuspension of an average size of 167 nm (NS-167 nm); 2) 90 minutes to prepare a nanosuspension of an average size of 400 nm (NS-400 nm); 3) 50 minutes to prepare a nanosuspension of an average size of 700 nm (NS-700 nm); and 4) 25 minutes to prepare a nanosuspension of an average size of 1700 nm (NS-1700 nm).
A reliable and reproducible HPLC assay was successfully developed for quantification of Mbz in aqueous solutions. The average retention time was 3.9 min and 8.4 min for the internal standard and Mbz, respectively. The standard curves, each containing eight points, were constructed at the linearity range of 0.025-10 μg/ml with correlation coefficients >0.999. The assay was validated with in-day variation (n=3) of 1.27% and between-day variation (n=6) of 2.77%. The mean linear regression equation of peak area ratio (Y) versus Mbz concentration (X) was Y=1.3412X+0.0042, which was used to determine Mbz concentrations in in vitro release studies and in cosolvent and nanoformulations prepared for in vitro and in vivo studies.
In Vitro Mbz Release from Cosolvent and 167 nm, 400 nm, 700 nm and 1700 nm Nanosuspensions in PBS
In vitro release studies of Mbz cosolvent and nanosuspensions with different sizes (NS-167 nm, NS-400 nm, NS-700 nm and NS-1700 nm) were performed in PBS with 0.2% Tween 80 at 37° C. (
Comparing the initial release rates of Mbz from different formulations in PBS/0.2% Tween 80, the Mbz nanosuspensions (<30%/hr) exhibited much slower initial release rates than the cosolvent formulation (115.66%/hr). Table VII shows the initial release rates (%/hr) of Mbz from cosolvent formulation, NS-167 nm, NS-400 nm, NS-700 nm and NS-1700 nm in PBS/0.2% Tween 80.
Among Mbz nanosuspensions of various sizes, the initial release rate of Mbz from the smallest size, NS-167 nm, was significantly faster than those from the large-sized nanosuspensions, NS-700 nm and NS-1700 nm at P<0.1. Although no statistical difference in the initial release rates was found among the rest groups, the mean values of the initial release rate were 27.14%/hr, 17.86%/hr, 11.83%/hr and 9.99%/hr for NS-167 nm, NS-400 nm, NS-700 nm and NS-1700 nm, respectively, decreasing as the particle size of Mbz nanosuspensions increased.
Comparing the release extents of Mbz from different formulations in PBS/0.2% Tween 80, the Mbz nanosuspensions exhibited a much lower release extents (<70% in 10 hr) than the cosolvent formulation (100% in 4.5 hr) (
These results suggest that Mbz nanosuspensions release drug much slower than Mbz cosolvent formulation. In addition, the nanosuspension of small size (NS-167 nm) show significantly faster initial release rates in PBS/0.2% Tween 80 than those from the large sized (NS-700 nm and NS-1700 nm) (P<0.1). The release extent of NS-167 nm and NS-400 nm was significantly larger than that of NS-1700 nm in 10 hr. Clearly, the particle size of Mbz nanosuspensions affects both of the in vitro release rate and release extent of the drug. The different release rate and extent of nanosuspensions of various sizes may be responsible for the effect on the pharmacokinetic properties of nanosuspensions.
In Vitro Mbz Release from Cosolvent and 167 nm, 400 nm, 700 nm and 1700 nm Nanosuspensions in Rat Plasma
In vitro releases of Mbz from cosolvent, nanosuspensions of different sizes (NS-167 nm, NS-400 nm, NS-700 nm and NS-1700 nm) were also evaluated in rat plasma at 37° C. (
Comparing the initial release rates of Mbz from different formulations in rat plasma, the Mbz nanosuspensions exhibited much slower initial release rates (<3.0%/hr) than the cosolvent formulation (33.45%/hr). Table IX lists the initial release rates (% hr) of Mbz from cosolvent formulation and the NS-167 nm, NS-400 nm, NS-700 nm, and NS-1700 nm nanosuspensions in rat plasma. Among Mbz nanosuspensions of various sizes, the initial release rate of Mbz from the smallest size, NS-167 nm (2.97%), was significantly faster than that from the largest size of nanosuspensions, NS-1700 nm (2.26%) at P<0.05. Although no statistical difference in the initial release rates was found among the rest groups, the mean values of the initial release rate were 2.97%/hr, 2.53%/hr, 2.47%/hr and 2.26%/hr for NS-167 nm, NS-400 nm, NS-700 nm and NS-1700 nm, respectively, decreasing as the particle size of Mbz nanosuspensions increased.
Comparing the release extent of Mbz from different formulations in rat plasma, the Mbz nanosuspensions exhibited a much lower release extent (<26% in 10 hr) than the cosolvent formulation (55% in 4 hr) (
These results suggest that Mbz nanosuspensions release drug much slower than Mbz cosolvent formulation in rat plasma. In addition, nanosuspensions of small size (NS-167 nm) show significantly faster initial release rates in rat plasma than that from the largest sized (NS-1700 nm) (P<0.05). The release extent of NS-167 nm was significantly larger than that of NS-1700 nm after 10 hr. Clearly, particle size of Mbz nanosuspensions affects both the in vitro release rate and release extent of the drug in rat plasma.
Comparing the release extent of Mbz from cosolvent and nanosuspensions in PBS/0.2% Tween 80 and rat plasma, the ratio of the release extent in these two release media in 4.5 hr was about 55% for cosolvent, and 40-50% in 48 hr for nanosuspensions. The significantly lower release extent in rat plasma than in PBS could be explained by plasma protein binding of Mbz. Mbz was considered as a high plasma-binding drug with about 90% of drug bound to human plasma protein. The initial release rates of Mbz from cosolvent and nanosuspensions in PBS/0.2% Tween 80 and rat plasma were also significantly different. The initial release rate in PBS was about 3.5 times of that in rat plasma for Mbz cosolvent and 4 to 9 times for Mbz nanosuspensions, suggesting that the plasma protein binding affect the initial release rate of Mbz cosolvent more than that of nanosuspensions. In short summary, the plasma protein binding of Mbz leads to much slower initial release rates and lower release extent of the drug from cosolvent and nanosuspensions in rat plasma than in PBS.
For quantification of Mbz in mouse plasma, the retention time was 3.9 min and 8.4 min for the internal standard and Mbz, respectively. No inference was found from blank mouse plasma. The calibration curves, each containing six concentration points, were constructed at the linearity range of 0.05-20 μg/ml with the correlation coefficients >0.999. The assay was validated with between-day variability (n=3) of 2.16%. Recovery of Mbz was about 95%. Table XI provides the calibration curve parameters for Mbz in mouse plasma samples. The mean linear regression equation of peak ratio (Y) versus Mbz concentration (X) was Y=0.6907X+0.045, which was used to determine Mbz concentration in plasma pharmacokinetic studies of Mbz in mice.
For quantification of Mbz in mouse six organs, brain, heart, kidneys, liver, lung, and spleen, the retention time was also around 3.9 min and 8.4 min for the internal standard and Mbz, respectively. No inference was found from six organ samples. The calibration curves were constructed at the linearity range of 0.05-10 μg/ml with the correlation coefficients >0.999 for all organs except for the spleen where the correlation coefficient is 0.9988. Each calibration curve contained six concentration points. The assay was validated for each organ with the between-day variability (n=3), which was the highest from the spleen (3.43%), the lowest from brain (1.03%), and similar from the rest organs (2.00%-2.75%). The recoveries of Mbz from the organs ranged from 80% to 87%. The heart had the highest recovery of 87.47%, followed by the brain (86.93%), the spleen (84.53%), the lung (82.58%), the liver (82.58%) and the kidneys (79.81%). All of these recoveries were acceptable. The mean linear regression equations of peak ratio (Y) versus Mbz concentration (X) were used to determine Mbz concentrations in respective organs in biodistribution studies of Mbz in mice (Table XII). Comparing the slopes of calibration curves from six organs, the brain and the heart had similar slopes around 0.37, while the rest organs had slopes around 0.33.
Plasma pharmacokinetics for Mbz from NS-167 nm and NS-1700 nm in mice have been studied. Both nanosuspension formulations exhibited sustained levels of Mbz plasma concentrations that were detectable up to 24 hr post dosing, compared to 6 hr for cosolvent (
From Table XIII, plasma pharmacokinetics were distinct between Mbz nanosuspensions and cosolvent. NS-167 nm and NS-1700 nm displayed a low Cmax/dose of 0.53 and 0.31 (mg/L)/(mg/kg), respectively, only ⅓ to ¼ of that from cosolvent [1.45 (mg/L)/(mg/kg)]. NS-167 nm and NS-1700 nm exhibited longer t1/2. α, and t1/2. β than cosolvent, particularly t1/2. β which was 10 to 12 times longer than that of cosolvent. In addition, V2 of NS-167 nm and NS-1700 nm were 0.79 L and 1.35 L, respectively, much larger than that of cosolvent. k21 for NS-167 nm and NS-1700 nm were 0.15 and 0.31 hr−1, respectively, 10 to 20 times slower than that of cosolvent (3.57 hr−1). Comparing NS-167 nm and NS-1700 nm, both exhibited low Cmax/dose, prolonged distribution and elimination half-lives and large Vss and V2. However, the Vss and V2 of NS-1700 nm were 1.45 and 1.35, respectively, apparently larger than those of NS-167 nm (0.85 and 0.79 L). The relative bioavailabilities were 0.48 and 0.39 for NS-167 nm and NS-1700 nm, respectively.
Different tissue distribution patterns between cosolvent and nanosuspension formulations were observed as anticipated from their plasma profiles (
Among organs, Mbz in cosolvent yielded the greatest exposure in liver with AUC0→∞ of 3.37 (hr*μg/g)/(mg/kg), followed by kidneys of 2.70 (hr*μg/g)/(mg/kg) and brain of 2.49 (hr*μg/g)/(mg/kg). However, for NS-167 nm, AUC0-48hr in liver was 136.15 (hr*μg/g)/(mg/kg), about 30 times of that from cosolvent, followed by those in spleen [83.30 (hr*μg/g)/(mg/kg)] and lung [22.04 (hr*μg/g)/(mg/kg)]. For NS-1700 nm, AUC0-24hr in liver was 116.60 (hr*μg/g)/(mg/kg), followed by that in lung [44.07 (hr*μg/g)/(mg/kg)]. The elimination half-lives of Mbz from the six organs after nanosuspension dosing ranged from 8 to 40 hours, substantially prolonged from 2 hours of Mbz from cosolvent. Comparing the organ pharmacokinetics between NS-167 nm and NS-1700 nm, the AUCs0-24 of NS-1700 nm in six organs except in liver were substantially larger than those of NS-167 nm. Furthermore, NS-1700 nm possessed longer t1/2 in lung and liver than NS-167 nm. Very high Mbz exposures in liver and spleen from nanosuspensions of two sizes were observed.
A size-dependant pharmacokinetic effect was observed with nanosuspension formulations of different size ranges of 138-1781 nm in rats (
The nanosuspension formulations of particle sizes >739 nm resulted in sustained levels of the drug for 48 to 96 hr, while plasma levels were detectable for only 48 hrs from formulations with size ≦253 nm. This was because of significantly shorter β half-lives for nanosuspensions with sizes ≦253 nm compared to those of formulations of size >739 nm.
To ensure the 14C-Mbz was homogenously present in the formulations, the content uniformity study was performed. The variations were 5.97% for cosolvent, 5.75% for NS-167 nm, 2.93% for NS-400 nm, 7.13% for NS-700 nm, 5.54% for NS-1700 nm (Table XV). One factor causing the variation may be due to the attachment of the formulation to the pipette tips in sampling.
To precisely measure the radioactivity, different amounts of rat organs (0.1 g, 0.2 g and 0.3 g) were tested for the quenching effect. The counting efficiencies were from 80% to 96% depending on the organs (Table XVI). The weight of 0.2 g was selected as the appropriate amount for the detection of radioactivity from rat organs due to the insignificant quenching effect. The counting efficiencies for 0.2 g of tissue were used to correct quenching effect in individual organs for rat biodistribution studies.
In the rat biodistribution studies, only a fraction of each organ (0.2 g) was taken for Mbz quantification. To ensure that the Mbz concentration measured from the fraction of organ can represent that in the whole organ, we have studied the homogeneity of 14C-Mbz in rat organs. To measure the variations among different sites of a particular organ, 200 mg of that organ was randomly taken from three sites and placed in each of three vials. The radioactivity from each vial was counted and the variation among three samples was calculated. As listed in Table XVII, the distribution variations were less than 8% for all organs, except that in heart, 14.02%. The variations were 3.01% for kidneys, 3.44% for liver, 5.15% for spleen, 5.67% for lung and 7.96% for brain.
Biodistribution Study of Mbz from Cosolvent and NS-167 nm, NS-400 nm, NS-700 nm and NS-1700 nm Nanosuspensions in Rats
Biodistributions of Mbz cosolvent and nanosuspensions (NS-167 nm, NS-400 nm, NS-700 nm and NS-1700 nm) in rats have been studied (
In
Different from cosolvent, Mbz nanosuspensions had very higher total drug concentrations in liver, spleen and lung, more than 8-fold of those in heart, kidneys and brain. The total drug concentrations in liver and spleen remained high at 48 hr. Mbz nanosuspensions exhibited longer half-lives of total drug in six organs than cosolvent (Table XVIII).
Comparing Mbz nanosuspensions of various sizes, the half-life of total Mbz in liver for NS-700 nm was longer than those for NS-167 nm and NS-400 nm. The half-life of total Mbz in spleen for NS-400 nm was longer than that for NS-167 nm. The half-life of total Mbz in liver for NS-1700 nm and in spleen for NS-700 nm and NS-1700 nm were not determined because the Mbz concentrations in these organs reached the highest value at 24 hr instead of a continuous decline from 5 min to 48 hr.
Total Mbz concentrations in organs from nanosuspensions of four sizes were compared at each time point (
Mbz nanosuspensions of the four sizes exhibited significantly high concentrations in blood than cosolvent at 5 min. The Mbz concentrations were detectable up to 48 hr for nanosuspensions, compared to being undetectable at 4 hr for cosolvent. NS-400 nm and NS-700 nm exhibited comparable Mbz concentrations in blood, which were significantly lower than that of NS-167 nm, but higher than that of NS-1700 nm. The Mbz concentrations in plasma at 5 min decreased when particle sizes increased.
Distribution Study of Parent Mbz from NS-167 nm. NS-400 nm. NS-700 nm and NS-1700 nm Nanosuspensions in Rat Liver and Spleen
Since very high total drug concentration in liver and spleen of rats were observed for Mbz nanosuspensions, the parent Mbz concentrations in these two organs were also evaluated by HPLC assay. The parent drug concentration of Mbz in liver and spleen after dosing was detectable up to 48 hours for all four sizes of nanosuspension. For NS-167 nm (
Mbz from cosolvent and nanosuspensions all followed a two-compartment model after i.v. injection. In both mice and rats, Mbz cosolvent and nanosuspension exhibited different pharmacokinetic properties. Mbz nanosuspensions had lower Cmax/dose, prolonged distribution and elimination half-lives and larger Vss and V2 compared with cosolvent (Table XIX). In both mice and rats, NS-1700 nm possessed lower Cmax/Dose, shorter t1/2. α, longer t1/2. β, and larger Vss and V2 than NS-167 nm. In addition, compared with NS-167 nm, the k10 of NS-1700 nm was slower, while the k12 and k21 were faster in both mice and rats (Table XIX). Therefore, the particle size of Mbz nanosuspensions showed similar effect on Mbz plasma pharmacokinetics in both mice and rats.
Mbz cosolvent and nanosuspensions exhibited similar distribution pattern differences in mice and rats. For Mbz cosolvent, liver had the highest drug concentration, followed kidney and other organs in both mice and rats (
The pharmacokinetic parameters derived from the liver and spleen profiles of Mbz were compiled in Table XX. Very similar Mbz concentration range was found in liver and spleen of mice than in those of rats. For NS-167 nm, the Mbz concentrations in the liver of mice were from 5.43 to 2.01 (μg/g)/(mg/kg), comparable to 4.30 to 1.56 (μg/g)/(mg/kg) found in the liver of rats. The Mbz concentration in the spleen of mice was from 3.26 to 0.91 (μg/g)/(mg/kg), comparable to 3.83 to 1.69 (μg/g)/(mg/kg) in the spleen of rats. Similarly, under the same dosing of NS-1700 nm, Mbz concentrations in the liver and spleen of mice were comparable to those in rats. However, the half-lives of Mbz in rat organs were longer than those in mouse organs. For NS-167 nm, the half-life of Mbz in the liver of rats was 32.66 hr, longer than that in mice (26.06 hr). The half-life of Mbz in the spleen of rats was 2.5 times of that in mice.
The allometric relationships between Mbz pharmacokinetic parameters (CL, Vss, t1/2, α, and t1/2, β) (Table XXI) and body weight for cosolvent, NS-167 nm and NS-1700 nm were plotted on a log-log scale (
The following references are cited herein.
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually incorporated by reference.
This is a continuation-in-part of pending nonprovisional application U.S. Ser. No. 10/640,467, filed Aug. 13, 2003.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10640467 | Aug 2003 | US |
| Child | 12220364 | US |
