Method of and compositions and sets of agents for enhancing oral biological availability of pharmaceutic agents

The invention relates to a use, kit and pharmaceutical composition for increasing the bioavailability of a taxane. More particularly, the invention relates to increasing the oral bioavailability of pharmaceutical agents that are poorly absorbed from the gastrointestinal tract and that allow the treatment of patients by oral administration of such agents. One aspect of the invention relates to the use of cyclosporins to enhance the oral bioavailability of paclitaxel and the corresponding taxanes.

2. Description of the prior art

Many valuable pharmacologically active compounds cannot be effectively administered by the oral route due to poor systemic absorption from the gastrointestinal tract. All of these pharmaceuticals are therefore generally administered by the intravenous or intramuscular route requiring the intervention of a physician or other healthcare professional, presenting severe discomfort and potential local shock.

188 281 for the patient, even requiring administration in a hospital setting, with surgical access, for certain IV infusions.

It has been contemplated that, in some cases, the poor oral bioavailability of drugs is due to the activity of a multidrug transporter - membrane-bound P-glycoprotein, which acts as an energy-dependent transport or efflux pump to reduce intracellular drug accumulation by forcing foreign substances out of the cell. This P-glycoprotein has been identified in normal tissues of the secretory endothelium, such as those that line the biliary tract, the brush border of the proximal tubule in the kidney and the lumen surface, and the vascular endothelial cells that line the blood-brain, placenta, and testes barrier.

The P-glycoprotein efflux pump is believed to prevent certain pharmaceutical compounds from crossing the cells of the mucosa of the small intestine and, therefore, from being absorbed into the circulation. Numerous known non-toxic pharmacological agents have been shown to inhibit P-glycoprotein, including, but not limited to, cyclosporin A (also known as cyclosporin), verapamil, tamoxifen, quinidine, and phenothiazines. Many of these studies have been aimed at achieving greater accumulation of cytotoxic drugs inside tumor cells. In fact, clinical experiments were conducted to investigate the effects of cyclosporin on the pharmacokinetics and toxicity of paclitaxel (Fisher et al., Proc.Am.Soc.Clm.Oncol., 13: 143, 1994); doxorubicin (Bartlett et al., J. Clin. One., 12: 835-842,1994); and etoposide (Lum et al., J.Clin.One., 10: 1635-42, 1992), all of which are anti-tumor agents known to be susceptible to multiple drug resistance (MDR). These experiments showed that patients receiving intravenous cyclosporine before or together with anti-cancer drugs had higher blood levels of these drugs, presumably due to reduced body clearance, and showed the expected toxicity at substantially lower dose levels. These findings aimed to establish that concomitant administration of cyclosporin inhibited the MDR effect of P-glycoprotein, allowing for higher intracellular accumulation of therapeutic agents. For a general discussion of the pharmacological implications of the clinical use of P-gp inhibitors, see Lum et al., Drug ResistClinOnc.Hemat 9: 319-336, (1995); Schinkel et al., Eur. J. Cancer. 31 A .: 1295-1298 (1995).

In the above studies on the use of cyclosporin to increase blood levels of pharmaceuticals that are susceptible to P-glycoprotein-mediated resistance, active agents, and cyclosporin were administered intravenously. It is not suggested in these publications that cyclosporine and other substances that inhibit the P-gp efflux pump could be administered orally to significantly increase the bioavailability of orally administered anti-cancer drugs and other pharmaceutical agents that are themselves poorly absorbed from the gut without producing highly toxic effects. side effects. Indeed, the 1995 review publication cited above, Lum et al., Showed that the co-administration of IV MDR inhibitors and chemotherapeutic agents subject to MDR increases the level of toxicity and worsens the severe side effects in patients. Schinkel et al. Briefly noted that MDR1 and P-glycoprotein are abundant in the intestinal mucosa and that this may affect the oral bioavailability of P-glycoprotein substrate drugs, but does not suggest or imply that oral administration of MDR inhibitors could improve the bioavailability of agents not available orally. Moreover, like Lum et al., Schinkel et al., Recalled that P-glycoprotein inhibitors can dramatically increase the toxicity of chemotherapy in patients and should therefore be used with caution.

In earlier publications, Schinkel et al. Showed that the absorption of orally ingested ivermectin was increased in mice homozygous for disruption of the MDR1 gene, compared to normal mice, demonstrating that P-glycoprotein plays a major role in reducing the bioavailability of this agent (Cell, 77 : 491-502,1994). In addition, these studies also showed that penetration of vinblastine into various tissues was enhanced in mutant mice.

None of the published studies have foreseen a course of action that would allow for effective oral administration of otherwise poorly bioavailable drugs, e.g. by determining appropriate dosage amounts and coordinating administration at specific times.

188 281 target drugs and bioavailability enhancers, that is, by demonstrating which MDR inhibitory agents are most suitable for promoting the oral absorption of each target drug or class of drugs.

The methods disclosed in the art to increase the intestinal absorption of drugs that have until now been administered only parenterally generally focus on the use of penetration and solubilization enhancers as stimulants, or the co-administration of P-glycoprotein inhibitors by perfusion into the lumen. the small intestine or by the intravenous route, e.g., Leu et al., Cancer Chemother.Pharmacol. 35: 432-436, 1995 (quinidine perfusion or IV infusion inhibits the efflux of etoposide from the blood into the lumen of the gastrointestinal tract). However, these methods suffer from numerous disadvantages. Solubility and permeation enhancers are often either impractical or ineffective when administered orally at the required dosages, and may impair the pharmacological activity of the target drug. Parenteral administration of P-gp inhibitors to humans at therapeutic (or near-therapeutic) doses may result in numerous clinical consequences. In the case of quinidine, e.g., IV administration can cause arrhythmias, peripheral vasodilation, gastrointestinal upset, and the like.

In PCT published application WO 95/20980 (published August 10, 1995), Benet et al. Discloses a significant method of increasing the bioavailability of orally administered hydrophobic pharmaceutical compounds. The method includes orally administering such compounds to a patient concurrently with a biamplifier, including an inhibitor of the cytochrome P450 3A enzyme, or a P-glycoprotein mediated membrane transport inhibitor. However, Benet et al., Did not actually provide a means of identifying which bioavailability enhancers would improve the availability of specific "target" pharmaceutical compounds, nor did they indicate specific dosage amounts, schedule or regimen for the administration of enhancers or target agents. In fact, although the Benet application lists dozens of potential enhancers (P450 3A inhibitors) and target drugs (P4503A substrates), the only combination of enhancer and target that is supported by any experimental fact in the application is ketoconazole as the enhancer and cyclosporin A as the target drug.

Describing the general characteristics of compounds that can be used as bio-enhancers by reducing P-glycoprotein transport activity, Benet et al. Indicate that they are hydrophobic compounds that generally, but not necessarily, contain two positively charged aromatic rings lying on a common plane, a group containing nitrogen or a carbonyl group - a class that includes a huge number of compounds, most of which would not provide the desired activity, absorption enhancer in the case of specific target agents. In addition, the classes of target agents described by Benet et al. Encompass the vast majority of pharmaceutical agents listed in the Physiciańs Desk Reference. These classifying criteria are of no value to practicing physicians seeking safe, practical, and effective means of orally administering specific pharmaceutical agents.

A further deficiency in Benet et al. Is the standard used to determine whether the bioavailability of a drug that is poorly absorbed when orally administered has improved. Benet et al. Indicate that any P-glycoprotein inhibitory agent which, when present in the intestine at a given concentration, reduces the transmembrane transport of rhodamine 123 by P-glycoprotein in the membrane of brush border vesicles or cells containing P-glycoprotein by 10% or more, can be considered a biostrengthening agent at this concentration and can be used in the practice of their invention. But increasing the absorption of only 10% from the intestine, an otherwise nonabsorbable agent, is inadequate to render the agent therapeutically valuable for any purpose. Indeed, according to the Federal Food and Drug Administration, two pharmaceutical preparations containing the same ingredient, differing in their bioavailability levels in the range of -20% / + 25%, are still considered bioequivalent, because for most drugs the difference in the concentration of the active ingredient in the blood within -20% / + 25% is not clinically significant. Approved Drug Products with Therapeutic Equivalence Ęyaluations (Dept. HHS, 14th Ed. 1994). When the FDA decides to take two preparations

188 281 pharmaceuticals are bioequivalent, doctors and pharmacists consider them to be freely replaceable with one another.

In general, Benet et al. Do not provide the knowledge that could be proceeded by a medical or pharmaceutical professional to identify an appropriate bio-enhancer / target drug combination, or to specifically design treatment regimens and plans that would make target agents therapeutically effective when administered. by mouth.

Thus, a safe, yet effective, method of increasing the systemic availability of orally administered drugs is desired that is currently only administered parenterally, as these are not absorbed sufficiently or properly when administered orally, and has not been provided in the prior art. .

Summary of the invention

Surprisingly, it has now been discovered and experimentally confirmed that certain agents which apparently inhibit P-glycoprotein drug transport activity, especially cyclosporin, can be used to substantially increase the oral bioavailability of otherwise poorly or unavailable pharmaceuticals, e.g. anti-cancer drugs such as paclitaxel (formerly known as taxol), as well as its analogs and derivatives, and etoposide.

The present invention relates, in one aspect, to a method of increasing the oral bioavailability of pharmaceutical agents that are poorly absorbed or not absorbed at all from the gastrointestinal tract or the intestine by administering pre-and / or concurrently to a subject by the oral route of one agent or combination of agents known to be effective in inhibiting drug transport through the P-glycoprotein pump If pretreatment is used, the bioavailability enhancer or agents must be administered in sufficient amounts and short enough time prior to administration of the drug for which the bioavailability is desired ("target drug" or "target agent") such that sufficient levels of the enhancer remain. at the site of absorption during administration of the target, effectively inhibiting the activity of P-glycoprotein or other transport substances for many drugs.

In a second aspect, the invention relates to compositions or dosage forms for the oral administration of pharmaceutical agents that until now have been available only by parenteral administration. A third aspect of the invention relates to the administration of such dosage forms or combinations thereof to patients for the treatment of conditions which are responsive to the active agents contained.

The invention also relates to pharmaceutical kits, comprising one or more oral dosage forms, containing the target agent, and one or more oral dosage forms, containing a booster.

Brief description of the drawings

Figure 1 is a graph reflecting the paclitaxel levels in serum samples taken over a period of 6-3 hours from three groups of rats: one group was given only intravenous paclitaxel, the other group was given only oral paclitaxel, and the third group was given oral paclitaxel and oral cyclosporin A (quoted as quoted above). hereafter as cyclosporine or CsA) in the doses before and immediately after the paclitaxel dose.

Figure 2 is a graph comparing serum paclitaxel levels from two of the three groups of rats reflected in Figure 1: the oral paclitaxel alone group and the oral paclitaxel group with the preceding and concurrent doses of oral cyclosporin.

Figure 3 is a graph reflecting the paclitaxel levels in plasma samples taken over a 24 hour period from two groups of rats: one group (A) was administered oral cyclosporin one hour before the combination of cyclosporin plus oral paclitaxel, and the other group (F) was administered oral cyclosporin alone. one hour before oral paclitaxel.

Figure 4 is a graph reflecting the paclitaxel levels in plasma samples from two groups of rats: one group (G) was administered paclitaxel IV, 3 hours after the oral dose of cyclosporin, and the other group (H) was administered paclitaxel IV only.

188 281

Figure 5 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a 24 hour period: one group (group A) received only radiolabeled IV paclitaxel, the other group (group B) received only radiolabeled paclitaxel orally, and the third group (group B). group (group C) was administered orally radiolabeled paclitaxel with an oral dose of cyicosporin one hour before and immediately after the dose of paclitaxel.

Figure 6 is a graph reflecting the levels of radioactivity detected in total blood samples collected from individual rats in group B (determined with reference to Figure 5).

Figure 7 is a graph reflecting the levels of radioactivity detected in total blood samples collected from individual rats in group C (determined with reference to Figure 5).

Figure 7A is a graph reflecting the total radioactivity levels of unchanged paclitaxel detected in total blood samples collected from a group of 10 rats over a 24 hour period, said group being administered orally radiolabelled paclitaxel (9 mg / kg) with doses of oral cyclosporin (5 mg / kg). ) one hour before and immediately after a dose of paclitaxel.

Figure 7B is a graph reflecting the levels of total radioactivity and paclitaxel metabolites 1, 2 and 3 detected in total blood samples from a group of 10 rats determined with reference to Figure 7A over a period of 24 hours.

Figure 8 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a period of 24 hours: one group was given 10 mg / kg orally verapamil as a booster, the other group was given oral progesterone as a booster, and a third group was given oral dipyridamole as a booster, with each group receiving an available dose of the same booster one hour later immediately after the oral dose of radiographically paclitaxel.

Figure 9 is a graph reflecting the levels of radioactivity detected in total blood samples collected over a 24 hour period from rats in the first group, defined with reference to Figure 8 (who were dosed orally with 10 mg / kg verapamil) treated with oral radiolabeled paclitaxel alone. and from the group of rats administered orally ciclosporin one hour before and again immediately after radiolabeled oral paclitaxel.

Figure 10 is a graph reflecting the levels of radioactivity detected in total blood samples collected over a 24 hour period from rats in the second group defined with reference to Figure 8 (orally administered progesterone), the orally radiolabeled paclitaxel alone and the rats group. who was administered orally ciclosporin one hour before, and again immediately after, radiolabeled oral paclitaxel.

Figure 11 is a graph reflecting the levels of radioactivity detected in total blood samples collected over a period of 24 hours from rats in the third group defined with reference to Figure 8 (which was administered orally with dipyridamole), the group of rats administered orally radiolabelled paclitaxel alone and from the group of rats. receiving oral ciclosporin one hour before and again immediately after radiolabeled oral paclitaxel.

Figure 12 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a 24 hour period: one group was orally administered 100 mg / kg verapamil¹ as a booster, the second group was orally administered megestrol acetate (commercialized as Megace® by Bristol-Myers Sauibb Oncology) as a tonic, and a third group was given oral ketoconazole as a booster, with each group receiving the same oral dose of the same booster, one one hour later, immediately after an oral dose of radiolabeled paclitaxel.

Figure 13 is a graph reflecting the levels of radioactivity detected in total blood samples taken over a period of 24 hours from the rats of the first group.

188 281 defined with reference to Figure 12 (which was administered orally with 100 mg / kg verapamil), the group administered orally radiolabeled paclitaxel alone, and the group of rats administered orally cyclosporin one hour before and again immediately after the radiolabeled oral paclitaxel.

Figure 14 is a graph reflecting the levels of radioactivity detected in total blood samples collected over a 24 hour period from the rats in the second group defined with reference to Figure 12 (orally dosed with megestrol acetate), the orally radiolabelled paclitaxel alone and groups ¹ As shown in Figure 12, the rats in the high dose verapamil group did not survive more than 8 hours.

rats dosed orally with ciclosporin one hour before and again immediately after radiolabeled oral paclitaxel.

Figure 15 is a graph reflecting the levels of radioactivity detected in total blood samples collected over a period of 24 hours from the rats in the third group defined with reference to Figure 12 (orally administered ketoconazole), the orally radiolabelled paclitaxel alone group and the rats treated with oral cyclosporin one hour before and again immediately after radiolabeled oral paclitaxel.

Figure 16 is a graph reflecting the levels of radioactivity detected in total blood samples collected over a 24 hour period from rats in the first group defined with reference to Figure 8 (to which 10 mg / kg verapamil orally was administered), the first group defined with reference to Fig. 12 (dosed orally with 100 mg / kg verapamil), the rat group receiving radiolabeled oral paclitaxel alone, and the rat group receiving oral ciclosporin one hour before and immediately after radiolabeled oral paclitaxel.

Figure 17 is a graph reflecting the levels of radioactivity detected in total blood samples collected over a 24 hour period from rats in the second group identified with reference to Figure 8 (which was orally administered progesterone), the second group identified with reference to Figure 8. 12 (orally dosed with megestrol acetate), the rats group that received radiolabeled oral paclitaxel alone, and the rats group that received oral ciclosporin one hour before and again immediately after radiolabeled oral paclitaxel.

Figure 17A is a graph reflecting a comparison of the dose response curves in the group of rats dosed orally with cyclosporin one hour before and again immediately after radiolabeled oral paclitaxel with the group of rats dosed with oral ketoconazole one hour before and again immediately after radiolabeled oral paclitaxel. Fig. 17B is a comparison of AUCQ0-24 values ​​determined in relation to the same two groups of rats.

Figure 18 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a 24 hour period: one group was given only radiolabeled etoposide IV, the other was given orally only radiolabeled etoposide, and the third was given orally radiolabeled etoposide with doses of oral cyclosporin before and immediately after the dose of etoposide, with an ordinate scale running from 0 to 1 in ppm of etoposide equivalents in total blood.

Figure 19 is a graph reflecting the levels of radioactivity detected in total blood samples collected from the three groups of rats defined with reference to Figure 18, with an ordinate scale ranging from 0 to 0.2 in ppm of radiolabeled etoposide equivalents in total blood.

Figure 20 is a graph reflecting the mean cumulative% dose of radioactivity detected in stool and urine of three groups of rats over a 168 hour period: one group was administered only radiolabeled IV paclitaxel, the other was administered only radiolabelled paclitaxel orally, and the third group was administered orally only radiolabeled paclitaxel with oral doses of ciclosporin before and immediately after the dose of paclitaxel.

188 281

Figure 21 is a graph reflecting the mean values ​​in ppm of paclitaxel equivalents detected in the blood and plasma of the three groups of rats defined with reference to Figure 20, 168 hours (7 days) after paclitaxel administration.

Figure 22 is a bar graph reflecting the mean values ​​in ppm of paclitaxel equivalents detected in various tissues (liver, kidney, testes and carcass) of the three groups of rats defined with reference to Figure 20, 168 hours (7 days) after paclitaxel administration.

Figure 23 is a bar graph reflecting the mean values ​​in ppm of paclitaxel equivalents detected in various tissues (muscle, pancreas, bone, lung and seminal vesicles) in the three groups of rats defined with reference to Figure 20, 168 hours (7 days) after administration of paclitaxel .

Figure 24 is a bar graph reflecting the mean values ​​in ppm of paclitaxel equivalents detected in various tissues (brain, heart, gastrointestinal tract, spleen and prostate) in the three groups of rats defined with reference to Figure 20, 168 hours (7 days) after administration of paclitaxel.

Figure 25 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a 24 hour period: one group was given orally cyclosporin D both one hour before and immediately after the oral dose of radiolabeled paclitaxel, the other group was given oral cyclosporin G both one hour before and immediately after the oral dose of radiolabeled paclitaxel, and the third group was given oral cyclosporin A for both one hour before and immediately after an oral dose of radiolabeled paclitaxel.

Figure 26 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a 24 hour period: one group was given oral ketoconazole both one hour before and immediately after the oral dose of radiolabeled paclitaxel, the other group was given a combined oral dose of cyclosporin A and ketoconazole both one hour before and immediately after the oral dose of radiolabeled paclitaxel, and the third group was given both cyclosporin A and both one hour before and immediately after an oral dose of radiolabeled paclitaxel.

Figure 27 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a period of 24 hours: one group was administered orally captopril both two hours before and immediately after the oral dose of radiolabeled paclitaxel, the other group was administered cyclosporin A both one hour before and immediately after the oral dose of radiolabeled paclitaxel, and the third group was administered orally with radiolabeled paclitaxel alone.

Figure 28 shows the radioactivity profile from HPLC plasma extract in rats in group C defined with reference to Figure 5.

Figure 29 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a period of 24 hours: one group was administered 10 mg / kg cyclosporin D orally one hour before and immediately after the oral dose of radiolabeled paclitaxel, the other group was administered 10 mg / kg cyclosporin F orally both one hour before and immediately after the oral dose of radiolabeled paclitaxel, the third group 5 mg / kg of cyclosporin D was administered both one hour before and immediately after the oral dose of radiolabeled paclitaxel, and the fourth group was administered 5 mg / kg cyclosporin F both one hour before and immediately after the oral dose of radiolabeled paclitaxel.

Figure 30 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a period of 24 hours: one (group A) was given only radiolabeled docetaxel ("Taxotere") IV, the other (group B) was given orally only radiolabeled docetaxel and the third group (group C) was given orally radiolabeled docetaxel with oral doses of cyclosporin one hour before and immediately after dose of docetaxel, with the ordinate of said plot running from 0-12.0 mean decetaxel equivalents in ppm.

188 281

Figure 31 is a graph reflecting the levels of radioactivity detected in total blood samples collected from the three groups of rats defined in Figure 30 but with the ordinate of said plot ranging from 0-2.0 mean docetaxel equivalents in ppm.

Figure 32 is a graph reflecting the levels of radioactivity detected in total blood samples collected from three groups of rats over a 24 hour period: one (group A) was administered only radiolabeled IV paclitaxel, the other (group B) was administered orally only radiolabeled paclitaxel, and the third group ( group C) orally administered radiolabeled paclitaxel with oral doses of cyclosporin one hour before and immediately after the dose of paclitaxel.

Figure 33 is a graph reflecting the levels of unchanged radiolabeled paclitaxel detected in total blood samples collected from the three groups of rats identified with reference to Figure 32 from 1-24 hours post-dose.

Figure 34 is a graph reflecting the levels of unchanged radiolabeled paclitaxel detected in total blood samples taken from 0-12 hours post-dose from the rats in group A defined with reference to Figure 32 and from the fourth group of rats (group D) administered with radiolabeled paclitaxel IV with oral doses of cyclosporin one hour before and immediately after the paclitaxel dose, with the ordinate of said plot running from 0-30 ppm paclitaxel.

Figure 35 is a graph reflecting the levels of unchanged radiolabeled paclitaxel detected in total blood samples drawn from 1-12 hours post-dose from the rats of group A defined with reference to Figure 32 and group D defined with reference to Figure 34, wherein the ordinate of said plot is from 0.000-5.000 ppm paclitaxel.

Figures 36-41 are schematic diagrams of the process of extracting and distributing radioactivity from a composite (homogenate) of various tissues of rats in groups A and C, respectively, as determined with reference to Figure 32.

Figure 42 is a graph reflecting the paclitaxel levels detected in plasma samples collected at the listed time intervals in a group of ten rats on the third and fourth days of a regimen in which they were administered twice daily an oral dose (5 mg / kg) of cyclosporin and, one hour later , a combination of the same dose of oral ciclosporin plus oral paclitaxel (3 mg / kg).

Detailed Description of the Invention

The present invention relates generally to increasing the oral absorption and bioavailability of orally administered pharmacologically active agents, especially agents that are poorly or not absorbed at all from the gastrointestinal tract or the intestine. The preferred embodiments of the invention relate to (a) a method of increasing the oral bioavailability of anti-cancer agents, in particular paclitaxel (currently commercialized as Taxol® by the Bristol-Myers Quibb Oncology Division) and its derivatives; other taxanes; the semisynthetic paclitaxel analog docetaxel (N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl paclitaxel), manufactured under the trade name Taxotere® by Rhone-Poulenc Rorer SA; and etoposide; (b) for dosage forms and kits for oral administration of antineoplastic agents and other drugs previously administered only parenterally.

The terms "oral bioavailability" and "oral bioavailability" as used herein refer to the systemic availability (ie, blood / plasma levels) of an administered amount of drug administered orally to a patient.

Paclitaxel is a natural diterpene product isolated from Pacific yew (Taxus breyifolia). It is a member of the taxane family of terpenes. It was first isolated in 1971 by Wani et al. (J.Am.Soc., 93: 2325, 1971), who characterized its structure by chemical methods and X-ray crystallography. One mechanism of its activity relates to the ability of paclitaxel to bind tubulin, and thus inhibit cancer cell growth. Schiff et al., Proc. Natl. Acad. Sci USA, 77: 1561-1565 (1980); Schiff et al., Nature, 277: 665-667 (1979); Kumar, J. Biol. Chem., 256: 10435-10441 (1981).

Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman et al., Yale Journal of Biołogy and Medicine, 64: 583, 1991; McGuire et al., Ann. Intem. Med., U_: 273). , 1989). It is effective

188 281 in chemotherapy for several types of cancer, including breast cancer (Holmes et al., J. Nat. Cancer Inst., 83: 1797, 1991), and has also been approved for the treatment of breast cancer. It is a potential candidate for the treatment of skin cancers (Einzig et al., Proc.Am.Soc.Clin.Oncol., 20:46) and head and neck cancers (Forastire et al., Sem.Oncol., 20:56, 1990). This compound is also potent in the treatment of polycystic kidney disease (Woo et al., Nature, 368: 750, 1994), lung cancer and malaria.

Paclitaxel is only slightly soluble in water and this poses problems in the development of suitable injectable and infusion formulations useful in anti-cancer chemotherapy. Some IV infusion paclitaxel formulations have been developed using Cremophor El ™ (polyethoxylated castor oil) as a drug carrier due to the water insolubility of paclitaxel. For example paclitaxel used in clinical trials under the aegis of the NCI was formulated in 50% Cremophor El ™ and 50% dehydrated alcohol. However, Cremophor El ™, if administered intravenously, is itself toxic and causes vasodilation, increased respiration, lethargy, hypotension and death in dogs. It is also thought to be responsible for the allergic-type reactions seen with paclitaxel administration.

In an attempt to increase the solubility of paclitaxel and develop safer clinical formulations, research was directed to synthesizing paclitaxel analogues where the 2 'and / or 7 positions were derivatized with groups that enhance water solubility. These efforts resulted in pro-drug compounds that are more water-soluble than the original compound, and that exhibit cytotoxic properties upon activation. One important group of such prodrugs includes the 2'-onium salts of paclitaxel and docetaxel, in particular the salts of 2'-methylpyridinium mesylate (2-MPM).

Paclitaxel is very poorly absorbed orally (less than 1%); see Eiseman et al., Second NCI Workshop on Taxol and Taxus (September 1992); Suffness et al., In Taxol Science and Appliccations (CRC Press 1995). Eiseman et al., Indicate that paclitaxel has a bioavailability of 0% when administered orally, and Suffness et al. reports that oral dosing with paclitaxel does not seem possible since no anti-tumor activity was found when administered orally up to 160 mg / kg / day. Moreover, an effective method of effectively administering paclitaxel orally (ie, a method of increasing the oral bioavailability of paclitaxel) or other oral taxanes or analogs of paclitaxel such as docetaxel, which have anti-tumor activity, has not been achieved. For this reason, paclitaxel has not yet been administered orally to humans, and certainly not in the treatment of paclitaxel responsive diseases.

Docetaxel becomes commercially available as Taxotere® parenterally for the treatment of breast cancer. To date, there is no mention in the scientific literature of the oral absorption of docetaxel in animals or patients.

Etoposide is a semi-synthetic derivative of podophyllotoxin and is used to treat certain neoplastic diseases, especially gamete cancers (e.g. nuclear carcinomas) and small cell lung cancers (Loehrer, Sem. Onc., 19 No. 6 supp.14, pp. 48-52, 1992). It is available as an oral dosage form (Vepesid® capsules, Bristol-Myers Sąuibb Oncology) but is not properly well absorbed orally (the mean value of oral bioavailability for etoposide capsules is close to 50%).

Cyclosporins are a group of nonpolar cyclic oligopeptides (some of which have immunosuppressive activity) produced by the genus Topycladium, including, for example, Topycladium Inflatum Gams (previously designated as Trichoderma polysporum), Topycladium terricola and other imperfect fungi. The major component, cyclosporin A (cyclosporin or CsA) has been identified in parallel with several other minor metabolites e.g. cyclosporins B through Z, several of which exhibit significantly less immunosuppressive activity than cyclosporin A. Numerous synthetic and semi-synthetic analogs have also been produced. See generally Jegorov et al., Phytochemistry, 38: 403-407 (1995). The present invention includes natural, semi-synthetic and synthetic analogs of cyclosporins.

Cyclosporins are neutral, lipophilic, cyclic undecapeptides with a molecular weight of about 1200. They are used intravenously or orally as immunosuppressants primarily for organ transplantation and some other purposes. Cyclosporins, especially

188 281 cyclosporin (cyclosporin A) are known inhibitors of the efflux pump of P-glycoprotein as well as certain P450-degrading enzymes, but to date, no effective ways to apply these properties clinically, in terms of clinical and commercial feasibility or regulatory approval have been developed.

Mechanically, orally administered cyclosporine has the ability to inhibit the P-glycoprotein pump in the upper small intestine, which is the absorption site for most drugs. With intravenous administration of a highly metabolized drug such as cyclosporine, it is not possible for it to arrive undamaged in that region of the gut where the drugs are normally absorbed. Following parenteral administration, ciclosporin is cleared by the liver and enters the bile and intestine distal from the area of ​​optimal absorption. One of the surprising findings of the invention is that the immunosuppression observed with certain cyclosporins is not inextricably linked to the improvement in the oral availability of the therapeutic agents. Thus, cyclosporin F enhances the oral bioavailability of paclitaxel even though, as reported in the literature, it does not exhibit immunosuppressive activity. Stewart et al., Transplantation Proceedings, 20: (supp. 3) 989-992 (1988); GranelliPipemo et al., Transplantation, 46: 53S-60S (1988).

Ketoconazole is a widely used antifungal imidazole derivative that has also been used in some areas of prostate cancer treatment. Ketoconazole, as one of its forms of action, has been shown to reverse MDR in highly resistant human KB carcinoma cells (Siegsmund et al., J. Urology. 151: 485-491, 1994), but can also inhibit cytochrome P-450 enzymes. metabolizing drugs.

It has now been found that many pharmaceutical agents with a poor oral absorption profile can be effectively administered orally with a systemic absorption sufficient to exhibit therapeutic activity levels when said agents are administered orally concurrently with an oral dose of certain cyclosporins or other agents known to inhibit multi-resistance. drugs, drug transport activity, intracellular P-glycoprotein pump, as well as certain enhancers, whose ability to inhibit P-glycoprotein transport has not yet been determined. A further surprising discovery of our invention is that under certain conditions, oral administration leads to a more favorable pharmacokinetic profile, better tissue penetration and a higher volume of distribution of the target therapeutic agent.

We have observed in animal studies that certain agents that inhibit multi-drug resistance, such as cyclosporine and ketoconazole, when administered orally immediately after and / or before a drug such as paclitaxel and etoposide, increase the absorption of the latter drugs from the gut in an unexpected manner. and the amazing degree that results in the achieved therapeutic levels. However, it is not at all obvious that these observed results are due to suppression of the P-glycoprotein pump

Another possible explanation for the observed increase in the bioavailability of paclitaxel and etoposide is that there may be an interaction at the level of drug metabolising enzymes for cyclosporin and paclitaxel. Both agents are known to be highly metabolized by the cytochrome P-450 system (e.g. P-450 3A), which is concentrated in the liver as well as in the small intestine. It is not excluded that the cyclosporin that was administered first might inhibit these enzymes so that paclitaxel, which is non-polished and lipophilic, might be absorbed. In the absence of this local inhibition, paclitaxel would be metabolized to more polar metabolites which would not pass through the mucosal cells. The inability to show the pharmacokinetic interaction between cyclosporine and paclitaxel when cyclosporin was administered 3 hours prior to paclitaxel IV administration suggested that the site of interaction was the lumen. Even this theoretical explanation does not explain our surprising finding that certain P-gp inhibitors (e.g. cyclosporins and ketoconazole) greatly increase the oral bioavailability of specific target drugs, while other agents known as active P-glycoprotein inhibitors show little activity as oral absorption enhancers for the same target drugs.

This theoretical inhibition of intestinal metabolism of the target agents would be less or not at all effective in increasing systemic blood levels when the target agent is administered intravenously. Moreover, because the primary effect of the oral absorption of the agent

188 281 of the enhancer may be a local effect in the intestinal lumen, sub-therapeutic doses should be effective in achieving the desired effect. This is an important consideration for enhancing agents such as cyclosporins, which have potent immunosuppressive activity and can present toxicity problems when administered at high doses. Our observation that non-immunosuppressive cyclosporins such as cyclosporin F may still function as oral enhancers has great clinical value.

It is noteworthy that while we have hypothesized about the mechanisms of action on which our invention is based, we hardly know the mechanism (s) responsible for the surprising discoveries discussed here; and it does not prevent those skilled in the art from practicing the described invention.

A method of increasing the oral bioavailability of a target therapeutic agent with poor oral bioavailability (average or mean bioavailability less than 50%), involves orally administering an agent that enhances oral absorption or bioavailability to mammalian patients (human or animal) concurrently, or before, or concurrently, and before. by oral administration to increase the amount and duration of absorption of the intact target into the bloodstream.

Orally administered enhancers that can be used in the present invention include, but are not limited to, the following:

Cyclosporins, including cyclosporins A to Z, but especially cyclosporin A (cyclosporin), cyclosporin F, cyclosporin D, dihydrocyclosporin A, dihydrocyclosporin C, acetylcyclosporin A, PSC-833, SDZ-NIM 811² (both from Sandoz Pharmaceutical Corp.), and the corresponding oligopeptides produced by species of the genus Topycladium. The structures of cyclosporin AZ are described in Table 1 below.

Antifungal agents - ketoconazole.

Cardiovascular drugs - MS-209 (from BASF), amiodarone, nifedipine, reserpine, quinidine, nicardipine, ethacrynic acid, propafenone, reserpine, amiloride.

Natural anti-migraine products - ergot alkaloids.

Antibiotics - cefoperazone, tetracycline, chloroquine, fosfomycin.

Antiparasitic agents - ivermectin.

Multidrug resistance reversal agents - VX-710 and VX-853 (Vertex Pharmaceutical Incorporated).

Tyrosine kinase inhibitors - genistein and the corresponding isoflavonoids, quercetin.

Protein kinase C inhibitors - calphostin.

Agents inducing apoptosis - ceramides.

Endorphin receptor active agents - morphine, morphine related compounds, other opioids and opioid antagonists including (but not limited to naloxone, naltrexone and nalmefene).

A class of orally administered target therapeutic agents whose oral absorption is enhanced ²SDZ-NIM 811 is a cyclosporin (Me-Ile-4), a cyclosporin antiviral non-immunosuppressive by enhancing agents including, but not limited to, the following agents:

Paclitaxel, other taxanes, docetaxel and derivatives and prodrugs of all in use, especially their 2'-MPM salts and other 2'-methylpyridim salts.

Other chemotherapeutic agents that have low or highly variable oral bioavailability, including etoposide, camptothecin, CPT-11 (Pharmacia and Upjohn), topetecan (SmithKline Beecham), doxorubicin, vincristine, daunorubicin, mitoxantrone, and colchicine, all of which are considered to be affected by P-glycoprotein efflux

Other drugs that have not been shown to be translocated by P-glycoprotein, but that can be made absorbable in the presence of a P-glycoprotein inhibitor in the gut, include ganciclovir, phoscamet, camptothecin, and camptothecin derivatives.

188 281

Table 1

Cyclosporin AZ

<img file="PL188281B1_D0001.tif" />

P-Ala CHCCHjJj

<td>Cyclo- dispute</td><td></td><td></td><td></td><td colspan="2">Amino acids</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>cy-</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td>S.</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td>

<td>CyA</td><td>Me bmt</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyB</td><td>Me bmt</td><td>AL a</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyC</td><td>Me bmt</td><td>Thr</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>Cyd</td><td>Me bmt</td><td>Val</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyE</td><td>Me bmt</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>vai</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>Va 1</td>

<td>Dig</td><td>Doks Me bmt</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>Me Leu</td><td>MeLeu</td><td>MeVa 1</td>

<td>CyG</td><td>Me bmt</td><td>Nva</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D ~ Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyH</td><td>Me bmt</td><td>Abu</td><td>Sar</td><td>Mel.eu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D ~ Ald</td><td>MeLeu</td><td>MeLeu</td><td>D-Mev</td>

<td>Cyl</td><td>Me bmt</td><td>Val</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>Leu</td><td></td>

<td>Tick</td><td>Doks Me bmt</td><td>Val</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyL</td><td>Bmt</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyM</td><td>Me bmt</td><td>Nva</td><td>Sar</td><td>MeLeu</td><td>val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>TIN</td><td>Me bmt</td><td>Nva</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>Leu</td><td>MeVal</td>

<td>CyO</td><td>Me Leu</td><td>Nva</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>Cyp</td><td>Bmt</td><td>Thr</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>cyQ</td><td>Me bmt</td><td>Abu</td><td>Sar</td><td>Val</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyR</td><td>Me bmt</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>Leu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>Leu</td><td>MeVal</td>

<td>CyS</td><td>Me bmt</td><td>Thr</td><td>Sar</td><td>Val</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>Cit</td><td>Me bmt</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>Leu</td><td>MeVal</td>

<td>CyU</td><td>My brother</td><td>Abu</td><td>Sa r</td><td>MeLeu</td><td>Val</td><td>Leu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>^cy^v</td><td>Me bmt</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyW</td><td>Me bmt</td><td>Thr</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>Val</td>

<td>CyX</td><td>Me bmt</td><td>Nva</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>Leu</td><td>MeLeu</td><td>MeVal</td>

<td>CyY</td><td>Me bmt</td><td>Nva</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>Leu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

<td>CyZ</td><td>Me Amx- nookies Lokwis</td><td>Abu</td><td>Sar</td><td>MeLeu</td><td>Val</td><td>MeLeu</td><td>Ala</td><td>D-Ala</td><td>MeLeu</td><td>MeLeu</td><td>MeVal</td>

188 281

The dosage amount of the enhancer co-administered with the target drug, in accordance with the invention, is about 0.1 to about 15 mg / kg of patient body weight. By "co-administration" of an enhancing agent is meant administration substantially co-administration with the target agent (or less than 0.5 hours before, less than 0.5 hours thereafter, or together), from about 0.5 to about 24 hours prior to administration of the agent. or both, i.e. one or more doses of the same or different enhancement agents, given at least 0.5 hours prior to, and one dose administered substantially simultaneously with (or together with, either immediately before or after) of the target agent. Additionally, by "co-administration" is meant the administration of more than one dose of the target within 24 hours after the dose of the enhancer, in other words, the enhancer (s) need not be re-administered before or at each administration.

The dosage amount of orally administered target agents will vary from drug to drug based on its therapeutic index, the requirements of the condition being treated, the status of the subject, and so on. The method according to the invention enables the oral administration of paclitaxel in an amount of about 20 mg / m2² up to about 1000 mg / m² (based on patient body surface area) or about 2-30 mg / kg (based on patient weight) in a single or divided daily dose (2-3), maintains plasma paclitaxel levels in humans in the range of 50-500 ng / ml for extended periods (e.g. 8-12 hours) after each oral dose. These levels are at least comparable to those achieved with 96-hour therapy with IV taxol infusion (which causes great inconvenience to the patient, discomfort, waste of time, possible infection, etc.). Moreover, such plasma levels of paclitaxel are more than sufficient to provide the desired pharmacological activities of the target drug, e.g. inhibition of tubulin degradation (which occurs at levels of about 0.1 μΜ, or about 85 ng / ml) and inhibition of protein isoprenylation (which occurs at levels of about 0.03 μΜ, or about 25 ng / ml), which is directly related to with its anti-tumor activity, by inhibiting the function of the oncogene and other signaling proteins which play a crucial role in the regulation of cell growth.

It may be appropriate in some cases to administer to a subject a higher aggravating starting dose of a target agent in order to achieve a peak blood level followed by lower maintenance doses.

Two or more different enhancers and / or two or more different target agents may be administered together, sequentially or intermittently, in all various aspects of the method of the invention.

The pharmaceutical composition of the invention can be used in a method of treating mammalian patients suffering from neoplastic tumors, tumors, Kaposi's sarcoma, malignant conditions, uncontrolled tissue or cellular proliferation secondary to damaged tissue, and any other disease state responsive to paclitaxel, taxanes, docetaxel. , etoposide, prodrugs and derivatives of all the former, paclitaxel 2'-MPM and docetaxel 2'-MpM, by orally administered dosage forms, containing one or more of these agents. The types of cancer that can be treated particularly effectively with oral paclitaxel, docetaxel, other taxanes, and their prodrugs and derivatives are hepatocellular carcinomas and hepatic metastases, and cancers of the gastrointestinal tract, pancreas and lungs. Examples of non-cancerous disease states that can be effectively treated with these orally administered active agents in accordance with the present invention are uncontrolled tissue and cell proliferation secondary to damaged tissue, polycystic kidney disease and malaria, including chloroquine and pyrimethamine resistant malaria parasites (Pouvelle et al., J.Clin. Invest. 44: 413-417,1994).

Antitumor agents which have so far been administered only parenterally can now be administered according to the invention by the oral route with sufficient bioavailability to ensure pharmacologically active blood concentrations, particularly effective in the treatment of patients with primary tumors and metastases. The active ingredients will penetrate the intestinal wall as a result of prior and / or simultaneous administration of MDR inhibitors or other enhancers, and will be rapidly taken up by the portal circulation, providing a higher local initial concentration of chemotherapeutic agents

188 281 in the liver (a far higher local concentration than currently achieved with IV infusion therapy) than in the general circulation, or in most other organs, for seven days. Furthermore, it should be noted that the higher hepatic levels of paclitaxel after oral administration may not reflect the increased plasma levels due to the high first hepatic pass-through effect. The method of selectively producing high blood levels of anti-tumor agents is particularly valuable in the treatment of liver cancers (e.g., liver cell cancer and liver metastases), gastro-elite cancers (e.g., colon, rectum), and lung cancers.

Similarly, after oral administration in accordance with the present invention, higher levels of paclitaxel are found at twenty four hours (using tissue separation analysis) in the gastrointestinal tract, pancreas and lungs, compared to systemic circulation and most other organs. This fact makes orally administered paclitaxel of great value in the treatment of gastrointestinal, pancreatic and lung cancers.

Figure 21-24 are particularly remarkable and surprising. Our invention, in some cases, provides a way to achieve comparable and sometimes higher local paclitaxel tissue concentrations via the oral route than the intravenous route. This is in line with the higher volume of distribution of the therapeutic agent. Moreover, it was found that oral administration of the enhancer before and immediately after the target agent (in the case of cyclosporin and paclitaxel, see Fig. 20) produces a higher concentration of the target agent in the urine even than IV administration. This should make the oral co-administration of the enhancer with the target the treatment of choice for patients with cancerous tumors or with urogenital metastases.

In addition to a higher local concentration of the active ingredients in the liver than previously achieved, the plasma and tissue distribution of the active target agent when administered orally with suitable enhancers as envisaged in the present invention is apparent and surprisingly similar to that observed with IV administration. A series of experimental animal studies showed that a stabilized state of plasma levels of paclitaxel was achieved with oral co-administration with CsA by the third day of the regimen. Target agent levels obtained at steady state were comparable to those obtained in patients with 96-hour IV infusion of paclitaxel. A 27% response rate was found in non-taxane metastatic breast cancer patients treated with a continuous 96-hour infusion every three weeks (Seidman et al., J.Clin.Oncol., 14: 1877, 1996). It is believed that similar results can be obtained with the treatment methods employed in the present invention without the discomfort, inconvenience, and risk of prolonging the IV infusion.

Besides, and quite significantly, the blood concentration in the clearance phase of paclitaxel and other anti-tumor agents mentioned above when administered orally as provided herein is approximately equal to that obtained with IV administration, and these higher, therapeutically effective levels can be maintained - 8-12 hours after each administration. The increase in urinary drug excretion after oral administration in the presence of CsA not only supports the enhanced oral absorption of paclitaxel, but also provides more drug delivery to the urogenital tract in the treatment of cancers.

Oral dosage forms of the target agents whose bioavailability is enhanced by co-administration of the enhancing agents may be the forms of conventional tablets, capsules, caplets, gelatin capsules, pills, liquids (e.g., solutions, suspensions, or elixirs), lozenges, and any other oral dosage form known in the pharmaceutical art. Liquid preparations may include, e.g. paclitaxel or other taxane on a vehicle containing Cremophor EI or other polyoxyethylene castor oil, alcohol, and / or polyoxyethylated sorbitan monoelate (e.g., Tween® 80, ICI Americas, Inc.). Each dosage form comprises an effective amount of the target agent (e.g., effective amounts of anti-tumor or anti-tumor, anti-tumor or anti-tumor agent) and inert pharmaceutically inert ingredients, e.g. conventional excipients, vehicles, fillers, binders, disintegrating agents, solvents, dissolving agents, sweetening agents, coloring agents and all other inactive ingredients,

188 281 which are regularly incorporated into pharmaceutical dosage forms for oral administration. Many such dosage forms and oral vehicles are listed in Reminpton's Pharmaceutical Sciences, 17th Edition (1985), right after the list of inactive ingredients. Each dosage form also contains a pharmacologically effective amount, e.g., an antitumor or tumor reducing effective amount of one of the target drugs.

The exact amounts of each of the target drugs in oral dosage forms will vary with the age, weight, disease, and condition of the patient. For example, paclitaxel dosage forms may contain sufficient amounts of paclitaxel to provide a daily dose of about 20-1000 mg / m2.² (based on patient body surface area) or 2-30 mg / kg (based on patient body weight) as single or divided (2-3) daily doses. Oral dosage forms of etoposide may contain sufficient amounts of etoposide to provide a daily dose of about 20-200 mg / m2 (based on average or mean patient body surface area) as single or divided (2-3) daily doses.

As already indicated, some target agents are commercially available in oral dosage forms despite their relatively poor or inadequate oral bioavailability. For example, Vepesid® capsules are available containing 50 mg of etoposide each.

In the established treatment regimen of an individual patient treated with an oral dosage form containing the target drug of the invention, it is necessary to take into account the increased bioavailability provided by the simultaneous and / or prior administration of enhancing agents. For example Although the manufacturer's recommended amount of Vepesid® capsules per dose for the treatment of small cell lung cancer is twice the IV dose rounded to the nearest 50 mg, the increased bioavailability of etoposide provided by prior and / or substantially concurrent administration of enhancing agents such as cyclosporin allows the much lower dosage of oral etoposide used to ensure the same effective blood levels of the drug, with greater penetration and stability of action and not increasing (and possibly reducing) toxic side effects. When administered orally, high peak levels that account for some toxicity can be avoided. Based on our experimental data (see fig. 18 and 19 and Table 6), which indicate that the oral absorption of etoposide is substantially complete (about 96%) in the presence of cyclosporin, the daily oral dose for etoposide in the treatment of testicular cancer should be about 50-100 mg / m2, and in the treatment of small cell lung cancer approximately 35-50 mg / m2, based on the patient's body surface area.

The dosing schedule for the treatment method used in the present invention, e.g., treatment of paclitaxel responsive diseases with paclitaxel oral dosage forms co-administered with boosting agents, can also be adjusted according to patient characteristics and disease state. The recommended dosing schedules for oral administration of paclitaxel are (a) a daily dosage, to a patient in need of it, of 1-3 equally divided doses, providing about 20-1000 mg / m2 (based on body surface area), with said daily dosing continued for 1 - 4 days in a row, every 2-3 weeks; or (b) administering about one day each week. The usual schedule equates to a 96-hour infusion of paclitaxel every 2-3 weeks, which some consider the recommended IV treatment regimen. The recommended dosing schedule for oral administration of etoposide, when co-administered with enhancers, is to administer to a patient in need 1-3 equally divided doses daily, providing about 50-100 mg / m2 (based on body surface area), for the treatment of cancer patients. testes and about 35-50 mg / m2 as a daily dose for the treatment of small cell lung cancer, with daily administration continued for 5-21 days in each case and with a period of 2-3 weeks between each treatment cycle.

Oral administration of potent chemotherapeutic agents in accordance with the invention can now reduce toxic side effects in many cases, compared to the current IV therapy. Absorption of the active agent through the intestinal wall (stimulated by enhancers), rather than the generation of sudden and rapid high levels in blood levels as is usually the case with IV infusion, ensures a more gradual reappearance of blood levels and a constant stabilization of these levels. levels at or near ideal size over a long period of time.

188 281

According to another aspect of the invention, a combination of oral dosage forms containing fixed amounts of at least one enhancing agent and at least one target agent is provided. For example such dosage forms may include tablets, capsules, caplets, gelatin capsules, pills, liquids, lozenges, and any other conventional oral dosage form containing, as active ingredients, an effective amount to enhance the oral bioavailability of an anti-tumor or anti-tumor agent, as well as suitable ingredients. inactive. One such combination product comprises from about 0.1 to about 15 mg / kg of one or more cyclosporins A, D, C, F, and G, dihydroCsA, dihydroCsC, and acetylCsA, together with about 20-1000 mg / m2 (based on 0 patient body surface area) paclitaxel, docetaxel, other taxanes or paclitaxel derivatives, or docetaxel such as paclitaxel 2'-MPM or docetaxel 2'-MPM. Another such dosage form comprises about 0.1 to about 15 mg / kg of cyclosporin or cyclosporin D or F, together with about 20 mg / m2 to 200 mg / m2 of etoposide.

Co-administration of enhancers with target drugs promotes not only the oral bioavailability of these agents, but also enables their use in the treatment of neoplastic tumors at sites highly protected by MDR, e.g. in the testes and brain. Another aspect of the present invention is therefore a method of delivering MDR-protected anti-tumor drugs to tumor sites by oral co-administration of enhancers and anti-tumor agents, which allows the treatment of brain tumors such as glioblastoma.

Yet another aspect of the present invention is a method of delivering an active metabolite of paclitaxel to diseased sites at therapeutic levels for the treatment of paclitaxel responsive diseases. In vivo major metabolites of paclitaxel have been identified, in particular the following metabolites of hydroxylated paclitaxel A, B and C:

<img file="PL188281B1_D0002.tif" />

A: R 1 = H, R 2 = OH; B: Ri = OH, R2 = H; C: Rr OH, R2 = OH (Paclitaxel: Ri = H, R2 = H)

It has been found in some in vivo tests that the metabolite B shown above (also reported in the literature as metabolite M4) has a higher therapeutic index (ratio of toxic concentration level to effective concentration level) than paclitaxel in some human tumor cell lines. The invention likely enables the delivery of enhancers of metabolite B and other active metabolites of paclitaxel to the tumor sites because when administered orally, all paclitaxel administered will pass through the liver and be metabolized by liver microsomes, delivering more of each metabolite to the circulation than is achieved with IV administration.

A further aspect of the invention relates to kits for use in the treatment of mammalian patients suffering from conditions responsive to any active target agent whose oral absorption and bioavailability is enhanced by the enhancer. These kits include one or more oral dosage forms, at least

188 281 of one enhancing agent and one or more oral dosage forms, of at least one target agent, or one or more dosage forms which consist of both.

By way of illustration, the kit of the invention may comprise one or more tablets, capsules, caplets, gelatin capsules or liquid formulations containing cyclosporin or ketoconazole, and one or more tablets, capsules, caplets, gelatin capsules or liquid formulations, containing paclitaxel or etoposide, in the dosage amounts within the ranges described above. Such kits may be used in hospitals, clinics, physicians 'offices, or patients' homes to facilitate the simultaneous administration of enhancement and target agents. The kits should also include attached printed information regarding the simultaneous administration of the enhancing agents and the target.

The subject kits can also include combinations of different enhancers and / or combinations of target agents. For example, the kit can contain oral dosage forms containing cyclosporin and ketoconazole as enhancers, respectively, with paclitaxel alone as the target or with a combination of paclitaxel and another anti-tumor drug. The second target should be a drug (such as paclitaxel) that exhibits poor oral bioavailability, but which, with co-administered enhancers, can achieve therapeutically effective blood levels when administered orally. The target agent can coexist with the enhancer in the same dosage form or it can be in a separate dosage form.

The following examples illustrate various aspects of the invention and demonstrate the unexpected, very significant increases in oral absorption of the target agents obtained. However, these examples are by no means intended to limit the invention or to cite specific enhancers or target agents, dosage amounts, testing procedures, or other parameters that must be used solely for the practice of the invention.

Example 1

Eighteen (18) healthy Sprague Dawley rats, all weighing between 225-275 grams and approximately six to eight weeks of age, were randomized into three groups of six animals. The first group of six rats received a single IV administration of paclitaxel at 9 mg / kg. The second group received a single oral dose of 9 mg / kg paclitaxel. The third group received a single oral dose of 5 mg / kg cyclosporin, and one hour later the same group received an oral dose of 5 mg / kg cyclosporin and 9 mg / kg paclitaxel.

Blood samples were taken from the tail vein of each rat at 0.5, 1, 2, 3, 4 and 6 hours after the paclitaxel dose. For the IV-treated rats of the first group, an additional blood sample was taken eight hours after the paclitaxel dose. Individual samples were centrifuged and the serum was separated. Six samples per group were combined for each time period to produce a single representative sample. All samples were assayed for unchanged paclitaxel by LC / MS with a lower limit of quantification of 50pg / ml.

The results of the study are shown graphically in Figures 1 and 2. Figure 1 compares all three groups of rats, while Figure 2 compares only the second and third groups that received oral paclitaxel. It can be seen that in the absence of cyclosporin, the serum bioavailability of paclitaxel was less than 1%, but increased to 6-7% in the third group that received cyclosporin one hour prior to the cyclosporin / paclitaxel combined dose.

The following table 2 lists the area under the curve (AUC) data determined for the three groups of rats. These data indicate that the AUC for six hours for the third group of rats receiving paclitaxel and cyclosporin together was nearly eight times the AUC of the second group of rats receiving oral paclitaxel only.

188 281

Table 2

The absolute bioavailability of paclitaxel

<td>AUC0-6 hrs ^IV (ngh / ml)</td><td>AUC0-6h PO (ng-h / ml)</td><td>Absolute F.</td>

<td> 9230*</td><td> 80</td><td> 0,9%</td>

<td colspan="2">* AUC value that does not include the 1-hour sample point</td><td></td>

<td>** F = (AUCpo / AUCiv] x100</td><td></td><td></td>

<td colspan="3">Interaction of paclitaxel with cyclosporine</td>

<td>AUC0-6h IV (ng-h / ml)</td><td>AUQ&gt; _6 h PO with cyclosporine (ngh / ml)</td><td>Relative F ***</td>

<td> 80</td><td> 629</td><td> 786%</td>

<td colspan="3">*** F = (AUCpoZ. Cyclosporin / AUCpo] x100</td>

Example 2

Forty (40) healthy Sprague Dowley rats with the same characteristics as those used in the study described in Example 1 were randomized into four groups of ten labeled groups A, F, G, and H. The following table 3 shows the treatments prescribed for each from the test groups and time periods for each dosing administration.

Table 3

<td>Group</td><td>Number of rats</td><td>Time (hour)</td><td>Treatment</td><td>Dose (mg / kg)</td><td>Way administration</td>

<td>AND</td><td> 10</td><td> 0</td><td>cyclosporine</td><td> 5</td><td>oral</td>

<td></td><td></td><td> 1</td><td>paclitaxel</td><td> 9</td><td>oral</td>

<td></td><td></td><td> 1</td><td>cyclosporine</td><td> 5</td><td>oral</td>

<td>F.</td><td> 10</td><td> 0</td><td>cyclosporine</td><td> 5</td><td>oral</td>

<td></td><td></td><td> 1</td><td>paclitaxel</td><td> 9</td><td>oral</td>

<td>G.</td><td> 10</td><td> 0</td><td>cyclosporine</td><td> 5</td><td></td>

<td></td><td></td><td> 3</td><td>paclitaxel</td><td> 9</td><td>IV</td>

<td>H.</td><td> 10</td><td> 0</td><td>paclitaxel</td><td> 9</td><td>IV</td>

Blood samples were taken from the tail vein of each rat at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 12 and 24 hours after paclitaxel administration. After the samples were treated appropriately and one pooled sample was made for each group, the plasma of each sample was determined for unchanged paclitaxel.

Figures 3 and 4 graphically illustrate the results of this study. Figure 3 shows a comparison between the concentration levels achieved over time in Group A, which received the preceding dose of cyclosporin and the paclitaxel-cyclosporin combined dose, one hour later, and Group F, which received the preceding dose of cyclosporin, followed by oral paclitaxel only, one hour later. one hour later. Fig. 4 reflects the comparison between

188 281 results in groups G and H, both of which received paclitaxel IV but group G received the preceding dose of oral cyclosporin three hours prior to paclitaxel. As indicated in Figure 4, the two groups showed substantially identical plasma levels of paclitaxel over the same time periods.

Table 4 reports the AUC data for the four groups of rats in this study. While the AUC values ​​for Group G and H were essentially the same, the AUC for Group A was 25-30% higher than that for Group F, indicating the importance of providing both pre-cyclosporin treatment and simultaneous administration of cyclosporin with paclitaxel.

Table 4

The bioavailability of paclitaxel in plasma

<td>Treatment</td><td></td><td>AUCo-,</td><td>F (%)</td>

<td>IV</td><td>(Group H)</td><td> 24280</td><td></td>

<td>IV + CsA available³</td><td>(Group G)</td><td> 24137</td><td> 99,4</td>

<td>Oral + CsA *</td><td>(Group F)</td><td> 1097</td><td> 4,5</td>

<td>Oral + CsA **</td><td>(Group A)</td><td> 1393</td><td> 5,7</td>

^and 3 hours before paclitaxel * 1 hour pre-treatment with CsA ** 1 hour pre-treatment and concurrent treatment with paclitaxel

Example 3

Eighteen (18) healthy Sprague Dowley rats with the same characteristics as those used in the study described in Example 1 were randomized into three groups of six rats, Groups A, B, and C. Group A was administered radiolabeled paclitaxel IV; group B received orally radiolabeled³H paclitaxel; and group C received an oral dose of cyclosporin followed one hour later by a combined oral dose of cyclosporin and radiolabeled oral paclitaxel.

Blood samples were taken from each rat's tail vein at the same times as described in Example 2. Samples were held as total blood. In addition, urine samples were collected from each rat, 4-24 hours after the paclitaxel dose. Blood and urine samples were analyzed for radioactivity.

A comparison of paclitaxel levels in the total blood samples of Groups A, B, and C is shown in Figure 5. Level comparisons for individual members of Groups B and C are shown in Figures 6 and 7, respectively.

In this study, the oral absorption of radioactivity (expressed as paclitaxel equivalent) in total blood was 10% in the absence of cyclosporin (Group B) and approximately 40% with concomitant administration of cyclosporin (Group C). This was determined by measuring the AUC of blood radioactivity following intravenous and oral radiolabeled paclitaxel. The bioavailability of paclitaxel has not been formally determined in this study as it would require unchanged drug determination at each time point. Nevertheless, at one point in time, the radioactivity was extracted from the plasma and, by standard HPLC, it was found that at least 32% of the radioactivity in the plasma was unchanged paclitaxel. The radioactivity profile of the HPLC plasma extract from Animal Group C, showing primarily one peak (which is paclitaxel), is shown in Figure 28. Table 5 below is a set of AUC, C data._mand_X'T_mand_X· And other data generated in this study.

188 281

Table 5

Total radioactivity for paclitaxel in blood / urine and% radioactivity extracted as paclitaxel in blood

<td>PK parameter</td><td>IV (A)</td><td>PO (B)</td><td>PO + CsA ** (C)</td>

<td>AUC0-24 (bg r ^ n.v. x h / ml)</td><td> 32,8</td><td> 3,3</td><td> 12,1</td>

<td>Cma (bg eq / ml)</td><td>ND</td><td> 0,21</td><td> 0,82</td>

<td>T._max (h)</td><td></td><td> 2</td><td> 5</td>

<td>% of dose in urine (4-24 hours)</td><td> 2,2</td><td> 1,9</td><td> 8,3</td>

<td>% paclitaxel *</td><td>ND</td><td> 7,8***</td><td> 32***</td>

*% as paclitaxel of extracted RA in a 4-hour sample ** CsA given 1 hour before and simultaneously with paclitaxel *** These numbers are bottom estimates based on an incomplete extraction procedure

Table 5A

Absorption of total radioactivity after oral administration³H-paclitaxel with / without cyclosporin (CsA) in rats (n = 10)

<td>PK parameter</td><td>Paclitaxel IV</td><td>Oral paclitaxel</td><td>Oral paclitaxel + + CsA</td>

<td>AUC0-24 hours (bg eq x hours / ml)</td><td> 23,8</td><td> 1,4</td><td> 8,1</td>

<td>AUC0— hr (bg eq x hr / ml)</td><td> 27,4</td><td> 4,5</td><td> 15,0</td>

<td>F (%) based on AUC 0.24 h</td><td></td><td> 5,9</td><td> 34,0</td>

<td>F (%) based on AUC0—</td><td></td><td> 16,4</td><td> 54,7</td>

Paclitaxel dose = 9 mg / kg

CsA (5 mg / kg 1 hour before and simultaneously with paclitaxel) F = AUCdj_Oral/ AUCiv

Table 5B

Oral pharmacokinetic parameters of paclitaxel with / without cyclosporin in rats (n = 10)

<td>PK parameter</td><td>Dose IV</td><td>PO dose</td><td>PO + CsA</td>

<td>AUC 0.24 h (bg h / ml)</td><td> 20,43</td><td> 0,314</td><td> 4,27</td>

<td>AUC ,, -— bg h / ml)</td><td> 21,02</td><td> 0,349</td><td> 5,41</td>

<td>F (%)</td><td></td><td> 1,700</td><td> 25,70</td>

<td>CL (ml / h / kg)</td><td> 29,00</td><td> 440,000</td><td> 430,00</td>

<td>V (ml / kg)</td><td> 4236,00</td><td> 5029,000</td><td> 5958,00</td>

<td>t1 / 2 (hours)</td><td> 6,80</td><td> 8,100</td><td> 9,60</td>

<td></td><td>(r2 = 0.96)</td><td>(r2 = 0.780)</td><td>(r2 = 0.95)</td>

CL = dose F * / AUC Dose = 9 mg / kg F = AUC_doustny / AUCiv

188 281

In rats that were treated as described in Example 3, the AUC for total radioactivity was determined. Based on the ratio of AUC_dosAt lower / AUCrv to infinity, oral absorption in the presence of ciclosporin increased to 54.7% compared with 16.4% in the absence of ciclosporin (Table 5A). Using a similar analysis for unchanged paclitaxel in the blood, the paclitaxel bioavailability was 25.7% in the presence of cyclosposrine and 1.7% in the absence of cyclosporine (Table 5b). Body clearance was surprisingly similar among the three treatment groups. The distribution volume of paclitaxel was increased by approximately 50% more in the cyclosporin and oral paclitaxel group compared to the paclitaxel IV group.

The following study design was used in Examples 4-5: Sprague-Dawley rats with the same characteristics as those in the study described in Example 1 were divided into three groups of three male rats each. All rats were fasted 12-14 hours prior to dosing. At the end of the fasting period, these rats receiving the enhancers were dosed with the boosters, and one hour later they were given a dose of radiolabeled (³H) paclitaxel (9 mg / kg) with simultaneous doses of a booster. Rats, not receiving boosters, were dosed with radiolabeled paclitaxel after fasting.

Blood was collected from each animal at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 12, and 24 hours following the paclitaxel dose. Urine was collected 4-24 hours post-dose. The total blood and urine radioactivity was then determined for each rat and the mean value for each group was calculated.

Example 4

Three groups of rats were administered as enhancers, respectively, 10 mg / kg verapamil orally, 5 mg / kg progesterone orally and 10 mg / kg dipyridamole, orally, both singly and with an oral dose of paclitaxel, one hour thereafter. A graphical comparison of the concentration profile in total blood over time (measured as concentration equivalents over time) determined for the three groups is shown in Figure 8. The data reflect roughly similar results using verapamil and dipyridamole as enhancers, with much lower bioavailability obtained with progesterone.

Figure 9 shows a graphical comparison between the concentration time profile of paclitaxel determined for the group of rats dosed with verapamil (10 mg / kg) as a booster, with the values ​​determined in the previous study for animals dosed with oral paclitaxel alone (9 mg / kg) and another group that was administered oral ciclosporin (5 mg / kg) both one hour before and immediately after an oral dose of paclitaxel (9 mg / kg). The cyclosporine group achieved far higher blood levels than the other groups during almost the entire 24 hour period.

Figures 10 and 11 show graphical comparisons analogous to Figure 9, but with the values ​​for the progesterone-administered group shown in Figure 10 and the dipyridamole group shown in Figure 11 in place of the verapamil group of Figure 9.

Example 5

Three groups of rats were administered as enhancers, respectively, 100 mg / kg verapamil orally, 5 mg / kg megestrol acetate orally and 50 mg / kg ketoconazole orally, both singly and with an oral dose of radiolabeled paclitaxel, one hour thereafter. Figure 12 shows a graphical comparison of the concentration profile in total blood over time (measured as concentration equivalents over time) determined for the three groups. The data reflect roughly similar results for verapamil and megestrol acetate as enhancers, with much higher bioavailability obtained with ketoconazole in the first 12 hours.

Figure 13 shows a graphical comparison between the concentration time profile of radioactivity determined for a group of rats that were administered verapamil as a booster (100 mg / kg) with the values ​​determined in a previous study for animals that were administered oral paclitaxel alone (9 mg / kg). and for another group of animals administered oral ciclosporin (5 mg / kg) both one hour before and immediately after the dose of oral radiolabeled paclitaxel (9 mg / kg).

Figures 14 and 15 show graphical comparisons analogous to Figure 13 but with the values ​​for the megestrol acetate treated group shown in Figure 14 and the ketoconazole group shown in Figure 15 in place of the verapamil group in Figure 13.

188 281

Figure 16 shows graphical comparisons between the concentration-time profiles of radioactivity determined for the group of rats dosed with 10 mg / kg verapamil in Example 4 and the group dosed with 100 mg / kg verapamil in Example 5.

Figure 17 shows a graphical comparison between the radioactivity concentration over time profiles determined for the group of rats dosed with 5 mg / kg progesterone in Example 4 and the group dosed with 5 mg / kg megestrol acetate in Example 5.

Both Figures 16 and 17 also show the same profiles reflected in Figures 13-15 for the study groups receiving radio-labeled oral paclitaxel alone and radio-labeled oral paclitaxel immediately after, and one hour at 5 mg / kg cyclosporin.

A dose-response data study was performed for ciclosporin. Increasing the dose to 10 mg / kg and 20 mg / kg one hour before and simultaneously with paclitaxel resulted in an oral absorption of radioactivity of approximately 45%. This contrasts with the findings for ketoconazole where doses up to 50 mg / kg were administered one hour prior to and concurrently with paclitaxel, and did not result in a further increase in oral radioactivity absorption (see Figures 17A and 17B).

The mean pharmacokinetic parameters for the study groups of animals discussed in Examples 4 and 5 are shown in Table 6.

The data generated by the studies of Examples 4 and 5 and reflected in Table 6 and Figures 8-17B clearly demonstrate the efficacy of cyclosporin as an oral bioavailability enhancer and its superiority over high or low dose verapamil, progesterone or megestrol acetate, especially in the first 12 hours. after the dose of paclitaxel. They also indicate that ketoconazole, although not as effective as cyclosporine, also has significant activity in promoting the oral absorption of paclitaxel.

³The study of example 4 is identified in table 6 as protocol NP951202 and the study of example 5 is designated as protocol NP960101.

Table 6

Mean pharmacological parameters for NP951202 and NP960101

<td>Protocol research</td><td>Treatment</td><td>Dose / route (mg / kg)</td><td>AUCo-24 (pg eq x hr / ml</td><td>F%</td><td>tl / 2 (hours)</td><td>Cmax (pg * eq / ml</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td>NP951OO1</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Only paclitaxel</td><td>9 / IV</td><td> 32,04</td><td></td><td> 20,15</td><td> 37,00</td>

<td></td><td>Only paclitaxel</td><td>9 / PO</td><td> 3,24</td><td> 10,1</td><td> 18,86</td><td> 0,21</td>

<td></td><td>Cyclosporine</td><td>5 / PO (c), 9 / PO (P) 5 / PO (C)</td><td> 12,02</td><td> 37,5</td><td> 14,51</td><td> 0,82</td>

<td>NP951202</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Verapamil</td><td>10 / PO (W), 9 / PO (P) 10 / PO (W)</td><td> 6,34</td><td> 19,8</td><td> 24,4</td><td> 0,78</td>

<td></td><td>Progesterone</td><td>5 / PO (pro), 9 / PO (P) 5 / PO (pro)</td><td> 3,78</td><td> 11,8</td><td> 20,0</td><td> 0,26</td>

188 281

continued from table 6

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td></td><td>Dipyridamole</td><td>10 / PO (D), 9 / PO (P) 10 / PO (D)</td><td> 6,18</td><td> 19,3</td><td> 26,6</td><td> 0,46</td>

<td>NP960101</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>* Verapamil (Animals pad-you)</td><td>100 / PO (W), 9 / PO (P) 100 / PO (W)</td><td>ON</td><td>ON</td><td>ON</td><td> 0,44</td>

<td></td><td>Megace</td><td>5 / PO (M), 9 / PO (P) 5 / PO (M)</td><td> 5,19</td><td> 16,2</td><td> 23,1</td><td> 0,44</td>

<td></td><td>Ketoconazole</td><td>50 / PO (K), 9 / PO (P) 50 / PO (KC)</td><td> 8,03</td><td> 25,1</td><td> 9,23</td><td> 0,69</td>

Example 6

Three groups of three male rats were each fasted 16-18 hours prior to dosing. At the end of the fasting period, one group of rats was administered an oral dose of 5 mg / kg cyclosporin. One hour later, this group was administered 5 mg / kg of cyclosporin orally with 1 mg / kg of radiolabel³H etoposide orally. Two other groups, after fasting, were administered only 1 mg / kg of etoposide, respectively³H IV and 1 mg / kg of etoposide H orally. The procedures for collecting blood and urine and determining total radioactivity were the same as in Examples 4 and 5, except that blood was collected at two additional times from the etoposide IV group at 0.033 and 0.25 hours. The obtained data are presented in Table 7.

Figures 18 and 19 graphically show the profile of the mean total blood concentration of etoposide over the time determined for the three treatment groups. In Fig. 18 the ordinate scale is from 0-1 etoposide equivalents concentration (ppm), while in Fig. 19 the ordinate scale is from 0-0.2 etoposide equivalents (ppm) to more clearly illustrate the differences between the values ​​obtained for the three groups. .

The data presented in Table 7 and Figures 18 and 19 show the effectiveness of cyclosporin as an enhancer of the oral bioavailability of etoposide, particularly in the first 12 hours after administration.

Table 7

Mean pharmacological parameters for NP960102

<td>Protocol research</td><td>Treatment</td><td>Dose / route (mg / kg)</td><td>AUCq_24 (pg eq x hr / ml</td><td>F%</td><td>t1 / 2 (hours)</td><td>Cmax (pg * eq / ml</td>

<td>NP960102</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Group A</td><td>Only etoposide</td><td>1 / IV</td><td> 1,08</td><td></td><td> 26,5</td><td> 2,16</td>

<td>Group B</td><td>Only etoposide</td><td>1 / PO</td><td> 0,61</td><td> 56,5</td><td> 19,1</td><td> 0,03</td>

<td>Group C.</td><td>CsA, etoposide + + CsA</td><td>5 / PO (C), 1 / PO (P) 5 / PO (C)</td><td> 1,04</td><td> 96,3</td><td> 18,1</td><td> 0,12</td>

188 281

Example 7

In another series of studies, three groups of three male rats each were fasted 16-18 hours prior to dosing. At the end of the fasting period, an oral dose of ketoconazole (2 mg / kg) was administered to one group of rats. One hour later, this group was orally administered 2 mg / kg ketoconazole with 1 mg / kg radiolabeled etoposide³H orally. The other two groups were treated in the same manner, except that they were administered 10 and 50 mg / kg of ketoconazole, respectively, after the fasting period, before and immediately after oral &amp; quot; H-etoposide. The procedures for collecting blood and determining total radioactivity were the same as in Examples 4 and 5. The data obtained is shown in Table 7A. Thus, unlike the effect of cyclosporine, which had a nearly doubled oral absorption of paclitaxel-derived radioactivity, ketoconazole administered over a wide dose range did not enhance the oral absorption of etoposide compared to etoposide alone.

Table 7A

<td>Treatment</td><td>Dose (mg / kg)</td><td>AUCq. 24 h (ggrównx hours / ml)</td><td>F%</td><td>Cmax ((ig * equiv / ml)</td><td>Tmax (h)</td><td>Tl / 2 (h)</td>

<td>NP960501</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Grp A Etoposide + Ketoconazole</td><td>I / EO (2 / Keto)</td><td> 0,54</td><td> 50,39</td><td> 0,026</td><td> 1</td><td> 47,8</td>

<td>Grp B Etoposide + Ketoconazole</td><td>I / EO (l 0 / Keto)</td><td> 0,69</td><td> 63,95</td><td> 0,032</td><td> 24</td><td> -91,5</td>

<td>Grp C Etoposide + Ketoconazole</td><td>I / EO (60 / Keto)</td><td> 0,64</td><td> 58,91</td><td> 0,060</td><td> 4</td><td> 38,1</td>

Example 8

Excretion equilibrium studies were performed with paclitaxel in rats. Three groups of 4-5 male rats each were fasted 12-14 hours prior to dosing. At the end of the fasting period, one group of rats was administered an oral dose of 5 mg / kg cyclosporin. One hour later, this group was orally administered 5 mg / kg cyclosporin with 9 mg / kg radiolabeled paclitaxel orally. The other two groups were administered 9 mg / kg radiolabeled IV paclitaxel and 9 mg / kg radiolabeled paclitaxel orally after the fasting period.

Urine and stools were collected from each animal in the following ranges: 0-2, 2-4, 4-8, 8-12, 12-24, 24-36, 36-48, 48-72, 72-96, 96-120 , 120-144, and 144-168 hours post-dose. Tissue harvesting was performed 168 hours post-dose. The procedure for determining total radioactivity was the same as in Examples 4 and 5. Fig. 20 shows a graphical comparison of the mean cumulative percentage of paclitaxel dose detected in the stools and urine of test animals over a 168 hour period. The group of rats administered cyclosporin with both pre- and oral paclitaxel showed a significantly lower percentage of the dose in the stool than the other two groups, and a markedly higher percentage of the dose in the urine, indicating that substantially more oral paclitaxel had passed through the intestinal wall and entered the system. the circulation of animals in the group that was treated with cyclosporine. In addition, the fact that the percentage of the dose in urine was significantly higher in the rats dosed orally with cyclosporin and paclitaxel as compared to the group dosed with paclitaxel IV indicates that co-administration resulted in a higher concentration of radioactivity passing through the urinary tract. sexual.

188 281

Figure 21-24 are bar graphs reflecting the mean values ​​in ppm of paclitaxel detected in various tissues excised from rats in the three study groups, group A representing paclitaxel IV treated animals, group B representing orally administered paclitaxel and group A C, representing the group treated with cyclosporin. These graphs show that the paclitaxel levels found in various tissues from the rats in group C were roughly comparable to the levels observed in the rats of group A that received paclitaxel IV, except in the liver, where the paclitaxel levels were more than twice as high in the group. cyclosporin treated group as in the paclitaxel IV administration group. The levels detected in the tissues of the B rats (which were administered oral paclitaxel alone) were quite low, in most cases far less than half the levels in both other groups.

The data obtained from this study are presented in Tables 8 and 9.

Table 8

Equilibrium excretion study for paclitaxel in rats Radioactivity in urine, stool and tissues as% of dose (mean values)

<td>A sample</td><td>Group A</td><td>Group B</td><td>Group C.</td>

<td>Urine</td><td> 9,160</td><td> 6,660</td><td> 18,350</td>

<td>Stool</td><td> 79,660</td><td> 84,410</td><td> 61,250</td>

<td>Tissues</td><td> 1,710</td><td> 0,600</td><td> 1,430</td>

<td>Whole</td><td> 90,530</td><td> 91,670</td><td> 81,030</td>

Table 9

Equilibrium excretion study for paclitaxel in rats Residual radioactivity in tissues expressed in ppm (mean values)

<td>A sample</td><td>Group A</td><td>Group B</td><td>Group C.</td>

<td>Brain</td><td> 0,101</td><td> 0,029</td><td> 0,096</td>

<td>Heart</td><td> 0,085</td><td> 0,025</td><td> 0,088</td>

<td>Lungs</td><td> 0,143</td><td> 0,030</td><td> 0,136</td>

<td>Liver</td><td> 0,237</td><td> 0,074</td><td> 0,566</td>

<td>The kidneys</td><td> 0,180</td><td> 0,032</td><td> 0,119</td>

<td>The muscles</td><td> 0,079</td><td> 0,025</td><td> 0,080</td>

<td>Gastrointestinal tract</td><td> 0,083</td><td> 0,021</td><td> 0,055</td>

<td>Testicles</td><td> 0,346</td><td> 0,037</td><td> 0,217</td>

<td>Three hundred</td><td> 0,078</td><td> 0,018</td><td> 0,080</td>

<td>Tushka</td><td> 0,143</td><td> 0,053</td><td> 0,099</td>

<td>Bone</td><td> 0,035</td><td> 0,007</td><td> 0,034</td>

<td>Spleen</td><td> 0,101</td><td> 0,024</td><td> 0,083</td>

<td>Prostate</td><td> 0,081</td><td> 0,022</td><td> 0,090</td>

<td>Seminal vesicles</td><td> 0,121</td><td> 0,024</td><td> 0,094</td>

<td>Blood</td><td> 0,112</td><td> 0,034</td><td> 0,106</td>

<td>Plasma</td><td> 0,126</td><td> 0,038</td><td> 0,124</td>

Example 9

Another tissue distribution study was performed with paclitaxel in rats. Two groups of 10 male rats each were fasted 12-14 hours prior to dosing. At the end of the fasting period, one group of rats was administered an oral dose of 5 mg / kg cyclosporin.

188 281

One hour later, this group was orally administered 5 mg / kg cyclosporin with 9 mg / kg radiolabeled paclitaxel orally. Another group was given only 9 mg / kg of radiolabeled IV paclitaxel after fasting.

Tissue harvesting was performed 24 hours post-dose. The procedure for determining total radioactivity was the same as in examples 4 and 5.

Table 9A reflects the values ​​in ppm of paclitaxel-derived radioactivity detected in various tissues excised from rats in the two treatment groups. One group representing animals administered paclitaxel IV and a second group representing those administered paclitaxel with cyclosporin administered 1 hour before and immediately after paclitakeel. The paclitaxel levels found in the various tissues of the cyclosporin-treated group of rats were roughly comparable to the levels observed in the paclitaxel IV-treated rats, with the exception of the spleen, pancreas and gastrointestinal tract where paclitakeel levels were approximately twice as high in the cyclosporin-treated group. as in the paclitaxel IV group.

A comparison of unchanged paclitaxel concentrations in different organs after paclitaxel IV alone versus oral paclitaSeel administered in the presence of cyclosporin is shown in Table 9B. Higher concentrations of unchanged paclitaxel after oral administration were found in the lung and gastrointestinal tract compared to the IV administration route.

Table 9A

Tissue ratio of paclitaxel equivalents ppm for groups C and A (average values)

<td>Tissue</td><td>Oral dose with CsA</td><td>Dose IV</td><td>Ratio</td>

<td>Brain</td><td> 0,267</td><td> 0,284</td><td> 0,94</td>

<td>Heart</td><td> 1,166</td><td> 0,576</td><td> 2,02</td>

<td>Lungs</td><td> 2,076</td><td> 1,230</td><td> 1,69</td>

<td>Liver</td><td> 4,328</td><td> 3,685</td><td> 1,17</td>

<td>The kidneys</td><td> 2,325</td><td> 1,259</td><td> 1,85</td>

<td>The muscles</td><td> 0,951</td><td> 0,639</td><td> 1,49</td>

<td>Gastrointestinal tract</td><td> 11,282</td><td> 5,673</td><td> 1,99</td>

<td>Testicles</td><td> 0,435</td><td> 0,804</td><td> 0,54</td>

<td>Pancreas</td><td> 1,999</td><td> 0,911</td><td> 2,19</td>

<td>Tushka</td><td> 1,043</td><td> 0,858</td><td> 1,22</td>

<td>Bone</td><td> 1,057</td><td> 0,612</td><td> 1,73</td>

<td>Spleen</td><td> 3,089</td><td> 1,180</td><td> 2,62</td>

<td>Prostate</td><td> 2,212</td><td> 1,660</td><td> 1,33</td>

<td>Seminal vesicles</td><td> 1,891</td><td> 2,693</td><td> 0,70</td>

<td>Blood</td><td> 0,373</td><td> 0,401</td><td> 0,93</td>

<td>Plasma</td><td> 0,370</td><td> 0,347</td><td> 1,07</td>

188 281

Table 9B

Removal of radioactivity from various tissues

<td>Group</td><td>Tissue</td><td>Ppm tissue 3H</td><td>% 3h characterized by HPLC</td><td>Paclitaxel tissue ppm</td><td>% 3h characterized as paclitaxel</td>

<td>IV</td><td>Liver</td><td> 3,7</td><td> 75,9</td><td> 1,34</td><td> 36,2</td>

<td></td><td>Lungs</td><td> 1,3</td><td> 79,5</td><td> 0,82</td><td> 63,1</td>

<td></td><td>Line stomach- -intestinal</td><td> 5,4</td><td> 78,1</td><td> 1,55</td><td> 28,7</td>

<td>Oral</td><td>Liver</td><td> 4,5</td><td> 75,5</td><td> 0,93</td><td> 20,7</td>

<td>with CsA</td><td>Phica</td><td> 2,3</td><td> 91,3</td><td> 1,42</td><td> 61,7</td>

<td></td><td>Line gastric- intestinal</td><td> 10,6</td><td> 91,4</td><td> 5,17</td><td> 48,8</td>

<td>Peak at 1.0 ppm</td><td>Liver</td><td> 1,0</td><td> 102,7</td><td> 0,77</td><td> 77,0</td>

Example 10

The procedure of Examples 4 and 5 was followed, but three groups of three males each were orally administered 5 mg / kg doses of cyclosporin D, cyclosporin G and cyclosporin A, both alone and one hour later, just after the oral dose of 9 mg / kg. kg of radiolabeled paclitaxel. Fig. 25 shows a graphical comparison of the concentration profiles in total blood over time for the radioactivity determined in these three test groups. Although all three cyclosporins showed essential activity in stimulating the oral absorption of paclitaxel, cyclosporin D, which had the lowest immunosuppressive activity (Jeffery, Clin. Biochem. 2A: 15-21 (1991)), of the three tested cyclosporins, showed the greatest bioavailability-enhancing activity.

Example 11

A number of studies were performed following the procedure used in Examples 4 and 5, and groups of three male rats each were dosed orally with 5-10 mg / kg of the various cyclosporins alone, then again one hour later, immediately after an oral dose of 9 mg / kg. kg of radiolabeled paclitaxel. Table 10 compares the AUC and% absorption from these studies, each identified by a protocol number beginning with "NP".

Table 10

AUC &amp;% of the absorption of various cyclosporins

<td>Protocol</td><td>Cyclosporine</td><td>Dose (mg / kg)</td><td>AUC0-24 (bg eq hr / ml)</td><td>% absorption</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td>NP960507</td><td>AND</td><td>2x5</td><td> 13,910</td><td> 42,1</td>

<td> 960503</td><td>AND</td><td>2x5</td><td> 10,17</td><td> 33,6</td>

<td> 960503</td><td>AND</td><td>2 x 10</td><td> 14,63</td><td> 48,3</td>

<td>NP960507</td><td>AcetylA</td><td>2x5</td><td> 8,39</td><td> 25,4</td>

<td> 960507</td><td>C.</td><td>2x5</td><td> 11,39</td><td> 34,5</td>

188 281

continued from Table 10

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td> 960507</td><td>E.</td><td>2x5</td><td> 5,96</td><td> 18,0</td>

<td> 960507</td><td>H.</td><td>2x5</td><td> 6,00</td><td> 18,1</td>

<td> 960507</td><td>AT</td><td>2x5</td><td> 5,02</td><td> 15,2</td>

<td>NP960103</td><td>D</td><td>2x5</td><td> 15,92</td><td> 48,2</td>

<td> 960103</td><td>G.</td><td>2x5</td><td> 13,22</td><td> 40,0</td>

<td>NP960704</td><td>D</td><td>2x 10</td><td> 14,23</td><td> 43,1</td>

<td> 960704</td><td>F.</td><td>2x 10</td><td> 11,99</td><td> 36,2</td>

<td>NP960605</td><td>F.</td><td>2x5</td><td> 8,99</td><td> 27,2</td>

<td> 960605</td><td>DihydroA</td><td>2x5</td><td> 8,5</td><td> 25,7</td>

<td>NP960801</td><td>Leu⁴</td><td>2x5</td><td> 7,38</td><td> 24,6</td>

<td> 960801</td><td>Dihydro C</td><td>2x5</td><td> 13,09</td><td> 45,1</td>

Example 12

The procedure of Examples 4 and 5 was followed, but three groups of three male rats each were dosed with 5 mg / kg cyclosporin A, 50 mg / kg ketoconazole and 5 mg / kg cyclosporin A plus 50 mg / kg ketoconazole, both alone and alone. and one hour later, immediately after an oral dose of 9 mg / kg of radiolabeled paclitaxel. A graphical comparison of the obtained results is shown in Fig. 26. The group receiving the combination of ketoconazole and cyclosporin A unexpectedly showed significantly higher levels of radioactivity in the blood over almost the entire 24 hour period than the groups receiving only one of these enhancers.

Example 13

The procedure of Examples 4 and 5 was followed, but three groups of three male rats each were dosed with 100 mg / kg captopril, both alone and two hours later, immediately following an oral dose of 9 mg / kg radiolabeled paclitaxel at 5 mg. / kg ciclosporin alone and again one hour later, immediately after an oral dose of 9 mg / kg of radiolabeled paclitaxin, and an oral dose of 9 mg / kg of radiolabeled paclitaxel alone. A graphical comparison of the obtained results is shown in Fig. 27.

The studies described so far have provided several previously unknown and unexpected discoveries, all of which are of great importance in the clinical management of many diseases, especially different types of cancers:

1. Certain MDR (P-glycoprotein) inhibitors, as well as other agents not known as MDR inhibitors, can be administered orally in order to effectively enhance the oral bioavailability of therapeutic agents that have so far been administered only parenterally because therapeutic blood levels cannot be obtained when administered oral.

2. Co-administration of the enhancers of the invention with target drugs having poor oral bioavailability may result in sustained blood levels of the target drugs, comparable to those obtained with IV infusions, but with less sudden initial increase in blood levels and hence less likely to occur. toxic side effects.

3. Oral co-administration of enhancers and target drugs increases the proportional concentration of the target agent in the liver, lung and gastrointestinal tract as compared to IV administration, making the new mode of administration particularly useful in the treatment of liver tumors and metastases.

4. Administration of the enhancer orally prior to the simultaneous administration of oral doses of the enhancer and the target drug increases the oral bioavailability of the drug

188 281 of the target to a significantly greater degree than the simultaneous administration of the enhancer and the target without prior administration of the enhancing agent. This results in plasma levels of the target drug reaching therapeutic levels.

5. Cyclosporins, such as cyclaspocin A, D, and F, are much more effective on Wednesdays for enhancing the bioavailability of anti-tumor agents than MDR inhibitors such as verapamil and progesterone.

Ketoconazole has clinically significant bioavailability enhancing activity, but less than that of ca'clocporinγ.

In general, the various aspects of the invention enable and are practical for the first time administration of an oral dosage form of widely used pharmaceuticals, particularly anti-cancer drugs such as paclitaxel related taxanes and etoposide, which until now could only be efficiently or reliably administered via IV infusions. The use of such oral dosage forms in the clinical management of cancer will increase patient comfort, convenience, consideration of wishes and safety, and will result in savings for patients, hospitals and government and private medical insurers.

In addition, the knowledge of the invention presented herein will provide information regarding target and enhancer selection as well as timing, scheduling, and dosing. This information and the methods and compositions of the invention will provide clinicians with procedures for determining therapeutic drug levels that require a narrow drug concentration window while avoiding unnecessary and often deleterious peaks and drops in blood concentration levels. In addition, the increased volume of separation of paclitaccel in the presence of cyclosporin suggests that more drug will be available for eliciting anti-tumor activity.

In addition to multi-drug resistance caused by the P-glycoprotein, encoded by the MDR1 gene, there is another recently discovered gene that confers a multi-drug resistance phenotype in certain laboratory systems: the multi-drug resistance protein gene, MRP (e.g., Zaman and in., Proc. Natl. Acad. Sci. USA. 91: 8822-8826,1994).

Less is known about this new gene and its 190 kd membrane-bound glycoprotein protein product. Although both the MRp and MDR1 genes encode membrane glycoproteins that can act as transporters for many drugs, there are differences in function, possibly substrates, and prognostic significance between the two genes. For example, expression of the MRP gene, but not of MDR1, is a good marker with a poor clinical outcome in patients with neuroblastoma. The purported function of MRP-related proteins is to serve as the efflux pump for glutathione S junctions. Thus, molecules that bind glutathione would be susceptible to the effects of the MRP-related system.

The oral bioavailability of pharmacologically active agents (or tumor exposure to such agents), which are resistant to MRP-associated proteins, may be enhanced by co-administered orally MRP inhibitors. A preferred embodiment for this method of increasing bioavailability is orally administering one or more MRP inhibitors prior to the simultaneous oral administration of the one or more MRP inhibitors and one or more target agents subject to MRP associated resistance.

Examples of this type of target include, but are not limited to, vinca alkaloids (e.g., vincristine), anthracyclines, epipodophyllotoccin (e.g., etoposide), and various taxanes. Examples of inhibitors that can increase the oral bioavailability of target agents include, but are not limited to, cyclosporins, ketoconazole, and the experimental drugs VX-710 and VX-853 (Vertex Pharmaceuticals, Inc., Cambridge, MA). The structures of VX-710 and VX853, as well as many related compounds, are disclosed in US Patent No. 5,192,773.

Another way to improve the oral bioavailability of agents subject to MRP-associated resistance is the co-administration of glutathione or substances that form glutathione conjugated products, which can interfere with the functioning of the MRp system and enhance intestinal absorption of the target, or increase the systemic exposure of the agents that undergo associated transport. with MRP.

188 281

Yet another system capable of conferring multi-drug resistance is the so-called pulmonary resistance associated protein (LRP), as it was first identified in a multi-drug resistant lung cancer cell line. This protein is the main structural protein of the so-called vault apparatus, a particle with a great abundance of cytoplasmic ribonucleins that got into humans from slimy molds. Inhibition of this system may also positively affect the oral bioavailability of certain agents. LRP is highly expressed in secretory and excretory epithelial cells as well as in cells chronically exposed to foreign substances such as cells lining the bronchi and intestines (Scheffer et al. Nature Medicine, 1: 578-582, 1955). Therefore, this system could also serve as a target for enhancing bioavailability.

It has therefore been shown that methods, compositions, and kits are provided that provide the various objects of the invention and that are well suited to the conditions of practical use.

As various possible embodiments of the above invention could be made, and as various changes could be made to the embodiments set forth above, it should be understood that all matters described herein are to be interpreted in an illustrative but not limiting sense.