JP2009538345A - 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin, its preparation and its use for the treatment of cancer - Google Patents

Abstract

More topoisomerase I than 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin or 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin in the diastereomeric mixture A better inhibitor, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereomer, is described. Also described are pharmaceutical compositions of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereomers and methods of using the compounds for inhibition of topoisomerase I and treatment of cancer. Is done.
[Selection figure] None

Description

Cross-reference to related patents and patent applications

  The subject of the present invention is US Pat. No. 6,177,439 issued Jan. 23, 2001, as well as filed on the same day as the present application, of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin. Related to co-owned U.S. non-provisional patent applications having the name of the crystalline form and having an agent case number of DRF 3.01-031 US, each of which is The entirety of which is hereby incorporated by reference.

  This application is a patent cooperation treaty application, Indian provisional application No. 897 / CHE / 2006 filed on May 24, 2006, and US provisional application No. 60 / 887,416 filed on January 31, 2007. And each of these applications is hereby incorporated by reference in its entirety as a part of this specification.

Field of Invention

  The present invention relates to the 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereomer of camptothecin and its use for the treatment of cancer.

Explanation of related technology

Camptothecin (CPT) is a pentacyclic plant alkaloid originally isolated from the Chinese tree Camptotheca acuminate by Wall et al. (Wall et al., J. Am. Chem. Soc, 88 : 3888 (1966)). The structure of camptothecin is shown in Formula I below.

  When unmodified, camptothecin is highly lipophilic and poorly water soluble. Initial clinical trials with camptothecin sodium solubilized in water with sodium hydroxide showed that the compound has antineoplastic activity, and further studies have shown that the activity is an inhibition of DNA topoisomerase I It was shown to be due to the activity of the compound as an agent. However, the therapeutic potential of camptothecin has not been well understood due to toxicity issues and limited water solubility.

  Attempts to provide improved properties for this compound included the stiffness and testing of many similar folds of camptothecin. For example, US Pat. No. 5,004,758 describes water-soluble camptothecin analogs and US Pat. No. 5,734,056 describes camptothecin analogs. Topotecan, a camptothecin analog, is discussed in US Pat. No. 5,004,758 and US Patent Publication 2007/0105885.

Various carbon 5-substituted analogs of 20 (S) -camptothecin are described in US Pat. No. 6,177,439. One such analog, 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin, has the structure shown in Formula II below.

  The 6,177,439 patent describes a mixture of diastereoisomers of this analog [where 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin, or 5- (Z-hydroxyethoxy) -20 (S) -referred to as camptothecin].

5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin is a 5-alkoxy substituted, 20 (S) -camptothecin analog with a pentacyclic structure. It has a chiral center at carbon 5 and carbon 20. The chiral center of carbon 20 corresponds to the natural S configuration. However, the carbon 20 substituents represent R and S diastereoisomers in approximately equal proportions. The molecular formula of 5 (RS)-(Z-hydroxyethoxy) -20 (S) -camptothecin is C ₂₂ H ₂₀ N ₂ O ₆ . The compound has a molecular weight of 408.41 g / mol and a melting point of 190 ° C. The diastereoisomeric mixture 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin or 5- (2′-hydroxyethoxy) -20 (S) -camptothecin has poor water solubility. Have.

The 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin diastereomer of 5 (RS)-(2'-hydroxyethoxy) -20 (S) -camptothecin is chemically 5 (S)-(2-hydroxyethoxy) -20 (S) -camptothecin, whereas 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin 5 (R )-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereomer is chemically described as 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin . 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is also chemically treated with 4- (S) -ethyl-4-hydroxy-12 (S)-(2-hydroxyethoxy) -1,12-dihydro-4H-2-oxa-6,12a-diazadibenzo [b, h] fluorene-3,13-dione is also described and has the chemical structure shown in Formula III below.

5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin has the chemical structure shown in Formula IV below.

  Generally speaking, isomers that are enantiomers, when present in a symmetric environment, are identical in chemical nature except that they have the ability to rotate plane polarized light in equal amounts and in opposite directions. And has physical properties. On the other hand, diastereomers (or diastereoisomers) are stereoisomers but not enantiomers. Diastereomers can have different physical properties and different reactivities, most often. In another definition, diastereomers are isomers that have opposite configurations at one or more chiral centers but are not mirror images of one another.

  Thus, it is clear from the above that it is desirable to provide the benefits of camptothecin in the treatment of cancer while reducing or avoiding one or more undesirable side effects or disadvantages that have so far limited its usefulness. .

  Briefly, therefore, the present invention provides a novel 5 (S)-(2 ′) that is substantially free of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereoisomers. -Hydroxyethoxy) -20 (S) -camptothecin directed to diastereoisomers.

  The present invention is also directed to a novel method for inhibiting the activity of topoisomerase I in a cell, said method comprising, against said cell, an effective amount of substantially 5 (R) − that inhibits topoisomerase I. Administration of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer without (2'-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer It is what.

  The present invention is also directed to a novel pharmaceutical composition comprising 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecinto as described above, and a pharmaceutically acceptable carrier. ing.

  The present invention also provides a novel method of using 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin for the manufacture of a pharmaceutical composition, wherein 5 (S)-(2 It is directed to a method comprising mixing '-hydroxyethoxy) -20 (S) -camptothecin with a pharmaceutically acceptable carrier.

The present invention is also directed to a method for producing the above 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin,
a) mixing 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin with a solvent selected from n-butanol or tetrahydrofuran;
b) refluxing the mixture for about 1 to about 4 hours;
c) cooling the mixture to a temperature of about 40 ° C. to about 50 ° C .;
d) separating the solid 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin from the mixture.

  The present invention is also directed to a novel method of using 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin to treat cancer in a patient, said method comprising said patient In contrast, the method comprises administering an effective amount of the 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin.

  Of the several benefits found to be achieved by the present invention, while providing the benefits of camptothecin in the treatment of cancer, while reducing one or more of the undesirable side effects or shortcomings that have limited its usefulness or The provision of methods and compositions to avoid is noted.

FIG. 1 shows an HPLC chromatogram of one sample of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin. FIG. 2 shows an HPLC chromatogram of another sample of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin.

Detailed Description of the Preferred Embodiment

  In accordance with the present invention, the 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer is obtained from the diastereoisomer mixture 5 (RS)-(2′-hydroxyethoxy) -20 ( It was found to be a significantly better topoisomerase I inhibitor than S) -camptothecin, or 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer.

  In fact, 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is more than 20 times stronger than 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin, and Almost 12 times stronger than 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin, a mixture of diastereoisomers.

  In addition, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereoisomers are obtained from the diastereoisomer mixture 5 (RS)-(2′-hydroxyethoxy) -20 ( It is shown to be a better inhibitor of tumor and cancer cell growth than S) -camptothecin, or 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer. Has been.

However, unexpectedly, the increased anticancer activity of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin diastereoisomers has a corresponding increase in toxicity to normal human cells. Not accompanied. In fact, 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin-5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer cancer cells When compared to its in vitro toxicity in healthy human cells, the safety margin (GI ₉₀ / GI ₅₀ ) is 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothe More than twice that of syndiastereoisomers.

  In addition, the presence of 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin diastereoisomers is indicated by the presence of 5 (S)-(2'-hydroxyethoxy) -20 (S) -campto Has a negative impact on the bioavailability of cecin diastereoisomers. Eventually, for the treatment of cancer, the 5 (S)-(2 '-(2'-(2'-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer free of 5 (R)-(2'-hydroxyethoxy) -20 Advantageously, the hydroxyethoxy) -20 (S) -camptothecin diastereoisomer is administered to the patient.

  For purposes of describing the present invention, certain terms are now defined as follows.

  The term `` 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin '' is the more active lactone form of 5 (S)-(2'-hydroxyethoxy) -20 (S)- It is intended to cover not only camptothecin but also the less active carboxylate form, including any salts formed therefrom.

  The term “inhibit” and variations thereof refer to any decrease in cellular activity, such as topoisomerase I activity, that can be measured via the methods described herein.

  The term “pharmaceutically acceptable” is defined in more detail below, but within the scope of appropriate medical judgment, without excessive toxicity, irritation, allergic response, etc., humans and lower animals Active agents, hydrates, solvates, salts suitable for use in contact with other tissues, corresponding to a reasonable benefit / risk ratio, and effective for the intended use Say, polymorphism, career, etc.

  The terms “solid”, “subject” and “patient” as used herein and defined in more detail below refer to any animal for which diagnosis, treatment or therapy is desired. As used herein, the term “animal” refers to humans and other animals, as well as other animals.

  As used herein, the terms “treating” and “treatment” of a condition, disorder, disease or condition are (1) affected by or predisposed to the condition, disorder, disease or condition Preventing or delaying the appearance of a clinical syndrome of the condition, disorder, disease or symptom occurring in a patient who has not experienced or presented with a clinical or subclinical syndrome of the condition, disorder, disease or symptom, (2 ) Inhibiting the condition, disorder, disease or symptom, ie stopping or reducing the occurrence of the disease or at least one clinical or subclinical syndrome thereof, or (3) alleviating the disease, That means causing a regression of the condition, disorder, disease or condition, or at least one regression of its clinical or subclinical preferences. . The benefit to the patient to be treated is statistically significant or at least recognizable by the patient and / or physician.

  The terms “effective amount” and “therapeutically effective amount” as used herein and defined in further detail below, are such as when administered to a patient to treat a condition, invitation, disease or condition. Refers to the amount of a compound sufficient to effect treatment. An effective or therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, physical condition and responsiveness of the individual to be treated.

  The terms “delivering” and “administering”, as used herein and defined in more detail below, provide a therapeutically effective amount of an active agent at one or more specific locations within a patient, It means producing a therapeutically effective concentration of the active agent in place. This can be accomplished, for example, by local or systemic administration of the active ingredient to a host, as described in more detail below.

  As used herein, the term “composition” means a product that contains a specific substance, as well as any product that results directly or indirectly from the combination of the specific components. A “pharmaceutical composition”, described in further detail below, comprises a combination of an active substance and one or more pharmaceutically acceptable carriers that are inert or active, the carrier comprising the composition in vivo. Making it suitable for in vitro or ex vivo diagnosis or treatment. The composition can also include stabilizers, preservatives, adjuvants, fillers, flavoring agents and other excipients.

  As used herein with respect to camptothecin, the term “carbon-5 position” refers to the 5th position of the chemical structure of 5 (S)-(2-hydroxyethoxy) -20 (S) -camptothecin shown in Formula II above. Means the carbon atom present, and the term "carbon-20 position" refers to the 20th position of the chemical structure of 5 (S)-(2-hydroxyethoxy) -20 (S) -camptothecin shown in Formula II above. Means a carbon atom present in

As used herein, throughout this description, chemically "5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin" refers to "4 (S) -ethyl-4- Hydroxy-12 (S)-(2-hydroxyethoxy) -1, 12-dihydro-4H-2-oxa-6, 12a-diaza-dibenzo [b, h] fluorin-3, 13-dione ” 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin ”.
As used herein, throughout this description, “5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin” is chemically referred to as “4 (S) -ethyl-4- Hydroxy-12 (R)-(2-hydroxyethoxy) -1, 12-dihydro-4H-2-oxa-6, 12a-diaza-benzo [b, h] fluorine-3, 13-dione " 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin ”.
As used herein, throughout this description, “5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin” is chemically referred to as “4 (S) -ethyl-4- Hydroxy-12 (RS)-(2-hydroxyethoxy) -1,12-dihydro-4H-2-oxa-6,12a-diaza-dibenzo [b, h] fluorine-3,13-dione " 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin ”or“ 5- (2′-hydroxyethoxy) -20 (S) -camptothecin ”.

  As used herein, the term “about” refers to the quantity measured, and the quality of measurement that would be expected by one of ordinary skill in the art to make a measurement and pay some level of attention depends on the purpose of the measurement and the accuracy of the measurement device. Appropriately, it means a normal variation in the measured quantity.

  The term “substantially free” is defined below.

  Example 26 of the '439 patent includes R- and S-diastereomers, namely 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (S)-(2'- The preparation of 5- (2′-hydroxyethoxy) -20 (S) -camptothecin, a mixture of hydroxyethoxy) -20 (S) -camptothecin, is described. This time, 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-(2 It has been discovered that it has certain unexpected and improved properties compared to '-hydroxyethoxy) -20 (S) -camptothecin. As briefly mentioned above, the inventors have shown that 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin exhibits, for example, toxicity, in vitro antitumor activity, bioavailability and topoisomerase I inhibition. It has been found that in several important properties, including the diastereomeric mixture 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin.

  In addition, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin, which is substantially free of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin, is a desirable anti-cancer agent. It was found to be a cancerous pharmaceutical compound.

  One aspect of the present invention is 5 (S)-(2′-hydroxyethoxy) -20 (S) substantially free of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin. -Camptothecin.

  When 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is said to be substantially free of 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin, it is Present in compounds containing both 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin The amount of (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin is present in the amount of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-( Meaning less than about 2% by weight of the total amount of 2'-hydroxyethoxy) -20 (S) -camptothecin. The amount of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin can be less than about 1.5% w / w, or can be less than about 1%, and about 0.5 % And even less than 0.1% w / w.

  In a variation of this aspect, the compounds of the invention are substantially optically pure. In one embodiment of this aspect, the compound has an overall chemical purity of greater than 98%.

もう一つの側面では、実質的に5(R)-(2'- ヒドロキシエトキシ)-20(S)-カンプトセシンを含有しない、5(S)-(2'-ヒドロキシエトキシ)-20(S)-カンプトセシンを調製する方法が提供される。この方法は、
ａ）5(S)-(2'-ヒドロキシエトキシ)-20(S)-カンプトセシンの溶解度の方が5(R)-(2'-ヒドロキシエトキシ)-20(S)-カンプトセシンの溶解度よりも大きい有機溶媒（n-ブタノールまたはテトラヒドロフランを使用することができる）中の、5(RS)-(2'- ヒドロキシエトキシ)-20(S)-カンプトセシンの溶液および／または懸濁液を提供する工程と；
ｂ）該溶液および／または懸濁液を加熱する工程と；
ｃ）該溶液および／または懸濁液を冷却して、沈殿を形成させることと；
ｄ）該沈殿を単離して、前記実質的に5(R)-(2'-ヒドロキシエトキシ)-20(S)-カンプトセシンを含まない5(S)-(2'-ヒドロキシエトキシ)-20(S)-カンプトセシンを得る工程
を含んでなるものである。 In another embodiment of the present invention, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin containing less than about 1.5% camptothecin heterodimer of formula V is Or less than about 1% heterodimer of formula V, or less than about 0.8% heterodimer, even less than about 0.5% heterodimer, and less than about 0.1% heterodimer. May be included, all of which are w / w based.

In another aspect, 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin-free 5 (S)-(2′-hydroxyethoxy) -20 (S) — Methods for preparing camptothecin are provided. This method
a) The solubility of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is greater than that of 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin Providing a solution and / or suspension of 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin in an organic solvent (n-butanol or tetrahydrofuran can be used); ;
b) heating the solution and / or suspension;
c) cooling the solution and / or suspension to form a precipitate;
d) isolating the precipitate to produce 5 (S)-(2′-hydroxyethoxy) -20 (5) substantially free of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin. Comprising the step of obtaining S) -camptothecin.

  The method may include a heating step comprising refluxing the solution and / or suspension. The method may further comprise refluxing the isolated precipitate with additional organic solvent.

In particular, the method for producing this 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is:
a) mixing 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin with a solvent selected from n-butanol or tetrahydrofuran;
b) refluxing the mixture for about 1 to about 4 hours;
c) cooling the mixture to a temperature of about 40 ° C. to about 50 ° C .;
d) separating the solid 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin from the mixture.

If a purer 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin product is desired, the method further comprises
e) washing the solid 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin with a solvent selected from n-butanol or tetrahydrofuran;
d) The solid 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is dried to give substantially 5 (R)-(2′-hydroxyethoxy) -20 (S) — A step of obtaining 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin free of camptothecin can be included.

  In another aspect, the present invention provides a method for inhibiting topoisomerase I activity in a cell, wherein the method substantially comprises 5 (R)-(2′-hydroxyethoxy) -20 (S) -Administration of an amount of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin free of camptothecin, said amount being sufficient to inhibit topoisomerase I ( A “topoisomerase I inhibitory effective amount”) method. The target cell can be cancer and administration can be performed in vitro or in vivo.

  The inventor has shown that 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin has several important properties including, for example, toxicity, in vivo antitumor activity, bioavailability and topoisomerase I inhibition. It was found that the diastereomeric mixture was different from 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin. Further, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin substantially free of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin is a desirable anti-cancer agent. It was also discovered that it is a cancer pharmaceutical compound.

  In the present invention, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is administered according to standard routes of drug delivery well known to those skilled in the art for the purpose of treating cancer.

  5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin can be supplied as a pure compound or in the form of a pharmaceutically active salt. Exemplary pharmaceutically acceptable salts include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, Pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, stearic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzene Prepared from sulfonic acid, pantothenic acid, toluenesulfonic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, cyclohexylaminesulfonic acid, ethanesulfonic acid, alginic acid, β-hydroxybutyric acid, galacturonic acid, and galacturonic acid.

  Suitable pharmaceutically acceptable base addition salts of the compounds of this invention include metal ion salts and organic ion salts. More preferred metal ion salts include suitable alkali metal (Group Ia) salts, alkaline earth metal (Group IIa) salts, and salts of other physiologically acceptable metal ions, It is not limited to these. Such salts can be made from ions of aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. Preferred organic salts can be prepared from tertiary amines and quaternary ammonium salts, some of which include triethylamine, diethylamine, N, N-dibenzylethylenediamine, chloroprocaine, colon, diethanolamine, ethylenediamine, meglumine (N- Methylglucamine) and procaine. All the above salts can be prepared by the skilled person from the corresponding compounds of the invention by conventional means.

The 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin of the present invention is used to form pharmaceutically acceptable carriers and excipients (herein “carriers”). And “excipient” can be described in any term). Pharmaceutically acceptable carriers and excipients include saline, Ringer's solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. It is not limited. The pharmaceutical composition may also contain stabilizers, antioxidants, colorants and diluents. Pharmaceutically acceptable carriers and additives are selected so that side effects from the pharmaceutical compound are minimized and to the extent that the properties of the compound are not offset or inhibited to the extent that treatment is ineffective. The

  In one embodiment of the invention, the pharmaceutical composition comprising 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is optionally 5 (R)-(2′-hydroxy Ethoxy) -20 (S) -camptothecin is substantially free.

  This pharmaceutical composition is typically 0.1-50% w / w, preferably 1-20% w / w of active 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin compound And the balance of the composition is a pharmaceutically acceptable carrier, diluent or solvent. Preferably, the pharmaceutical composition is in unit dosage form containing a suitable amount of the active compound, eg, an effective amount of the active compound to achieve the desired purpose.

  The pharmaceutical composition of the present invention may be administered enterally and / or parenterally. Oral (intragastric) administration is the preferred route of administration. Pharmaceutically acceptable carriers for the methods of the present invention can be in solid dosage forms including tablets, capsules, pills, and granules, which are coatings and shells such as enteric well known in the art. It can be prepared with a coating or the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.

  Parenteral administration includes subcutaneous administration, intramuscular administration, intradermal administration, intramammary administration, intravenous administration, and other methods of administration known to those skilled in the art. Enteral administration includes solutions, tablets, sustained release capsules, enteric coated capsules, and syrups. When administered, the pharmaceutical composition may be at or near body temperature.

  Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may be formulated as pharmaceutically elegant and palatable formulations. To provide one or more substances selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients include inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents (eg corn starch or alginic acid) binders (eg starch, gelatin Or acacia), and lubricants (eg, magnesium stearate, stearic acid or talc). The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be used.

  Formulations for oral use are also hard gelatin capsules in which 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin Alternatively, it may be presented as a soft gelatin capsule in which the active ingredient is present as such or mixed with water or an oily medium such as peanut oil, liquid paraffin or olive oil.

  Aqueous suspensions containing 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin in a mixture with excipients suitable for the manufacture of aqueous suspensions can be prepared . Such excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia; dispersants or wetting agents are naturally occurring Phosphatides (eg lecithin), condensation products of alkylene oxide and fatty acids (eg polyoxyethylene stearate), or condensation products of ethylene oxide and long chain fatty alcohols (eg heptadecaethyleneoxycetanol), or ethylene oxide and fatty acids And condensation products with partial esters derived from hexitol (for example, polyoxyethylene sorbitol monooleate), partial esters derived from ethylene oxide and fatty acids and hexitol anhydrides It may be a condensation product with tellurium (eg, polyoxyethylene sorbitan monooleate).

  The aqueous suspension may also contain one or more preservatives (eg, ethyl p-hydroxybenzoate or n-propyl), one or more colorants, one or more flavoring agents, or one or more sweetening agents (eg, sucrose or saccharin). ) May be contained.

  Oily suspensions include 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin, omega-3 fatty acids, vegetable oils (eg, acacia oil, olive oil, sesame oil, or coconut oil), or mineral oil ( For example, it may be formulated by suspending in liquid paraffin). The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

  Dispersible powders and granules suitable for preparation of an aqueous suspension can be obtained by adding 5 (S)-(2 ′ in a mixture with a dispersing or wetting agent, suspending agent and one or more preservatives by the addition of water. -Hydroxyethoxy) -20 (S) -camptothecin. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavoring and coloring agents may also be present.

  5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin and compositions comprising it may be administered parenterally, subcutaneously, or intravenously, or intramuscularly, or It can also be administered by infusion techniques either intrasternally or in the form of a sterile injectable aqueous or collagen suspension. Such suspensions may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above, or other acceptable substances. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable carriers and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, n-3 polyunsaturated fatty acids may be used in the preparation of injectable formulations.

  Administration can also be by inhalation in the form of an aerosol or solution for nebulizers, or the drug in a suitable nonirritating excipient (which is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum. Can also be done rectally in the form of suppositories prepared by mixing with the drug. Such excipient materials are cocoa butter and polyethylene glycols.

  Buccal or “sublingual” administration is also encompassed by the present invention, including lozenges or chewable gums containing the compounds described herein. The compounds can be deposited in flavored substrates, usually sucrose and acacia or tragacanth, and flavors containing the compounds in inert substrates such as gelatin and glycerin or sucrose and acacia. It can be a lock.

  Topical delivery systems are also encompassed by the present invention and include ointments, powders, sprays, creams, gels, eye washes, solutions or suspensions.

  Powders have the advantage that they can stick to moist surfaces and consequently remain active for a long time. Thus, the powder is particularly attractive for treating cancer, such as cancer in the ear canal. For the same reason, creams are also effective pharmaceutically acceptable carriers.

  The compositions of the present invention can optionally be supplemented with additional agents such as, for example, viscosity improvers, preservatives, surfactants and penetration enhancers.

  Such viscosity generating agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, or other agents known to those skilled in the art. Such agents are typically used at a level of 0.01% to 2% by weight.

  Preservatives are optionally used to prevent microbial contamination during use. Suitable preservatives include polyquaternium-1, polybenzarnium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, edetate disodium, sorbic acid or known to those skilled in the art Other drugs are included. The use of polyquaternium-1 as an antibacterial agent is preferred. Typically, such preservatives are used at a level of 0.001% to 1.0% by weight.

  The solubility of the components of the composition of the present invention may be improved by surfactants or other suitable co-solvents in the composition. Such cosolvents include polysorbates 20, 60 and 80, polyoxyethylene / polyoxypropylene surfactants (eg pluronic F-68, F-84 and P-103), cyclodextrins, or known to those skilled in the art Other drugs are included. Typically, such cosolvents are used at a level of 0.01% to 2% by weight.

  Pharmaceutically acceptable excipients and carriers include all of the above. The above considerations regarding effective formulations and methods of administration are well known to those skilled in the art and are described in standard textbooks. For example, Gennaro, AR, Remington: The Science and Practice of Pharmacy, 20th Edition, (Lippincott, Williams and Wilkins), 2000; Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pennsylvania, 1975; Liberman, See et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999. I want to be.

  For purposes of the present invention, it is preferred that the amount of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin comprises an effective amount of the compound. Accordingly, the present invention encompasses methods of preventing or treating cancer, neoplasia and / or neoplasia-related diseases or disorders (herein collectively referred to as “cancer”) Patients in need of such prophylaxis or treatment, and the method provides the patient with an effective amount of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin as described herein. Administering.

Effective amount or dose of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin /
Many factors are considered by the diagnostic physician in determining the ability and duration of action of the compound used, the nature and severity of the disease to be treated, and the gender of the patient being treated, This includes but is not limited to age, weight, general health status, individual responsiveness, and other related situations. The amount of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin required for use in the treatment or prevention of cancer varies widely, and is individual for each particular case. Will be adjusted to meet requirements.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture or in animal models. A therapeutically effective dose refers to that amount of active compound that ameliorates the symptoms or its syndromes. The therapeutic effect and toxicity in the cell culture or animal model may be determined by standard methods (eg, ED ₅₀ : therapeutically effective dose in 50% of the population; LD ₅₀ : lethal in 50% of the population Dose). Data obtained from cell cultures and animal models can then be used to formulate a dosage range of the compound for use in a patient.

  In general, suitable daily dosages are described herein for administration to adults, but if this is advisable, the limits specified as preferred may be exceeded. The daily dose can be administered as a single dose or as a divided dose. Those skilled in the art will understand that the dosage may be determined using guidelines from the literature [Goodman & Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711]. . Typical recommended dosage regimens are generally about 0.01 mg / kg / day to about 50 mg / kg / day, or about 0.05 mg / kg / day to about 25 mg / kg / day, or about 0.1 mg / kg / day to about 10 mg / kg / day, or about 0.1 mg / kg / day to about 5 mg / kg / day, or even about 0.2 mg / kg / day to about 3 mg / kg / day It will span.

  In one embodiment, the crystalline 5 (S) -CPT is about 1 mg to about 1000 mg per capsule, or about 2 mg to about 500 mg or about 5 mg to about 250 mg of 5 (S) per capsule. It can be provided as a hard gelatin capsule for oral administration containing 5 (S) -CPT of -CPT. When the patient to whom 5 (S) -CPT is administered is a human adult with an average body weight of about 70 kg, the daily dose of 5 (S) -CPT is about 0.01 mg / kg / day to about 50 mg / kg / day, or about 0.05 mg / kg / day to about 25 mg / kg / day, or about 0.1 mg / kg / day to about 10 mg / kg / day, or about 0.1 mg / kg / day to about 5 mg / kg / day, or even about 0.2 mg / kg / day to about 3 mg / kg / day.

  As used herein, the term “patient” for therapeutic purposes includes any patient and can be a patient in need of treatment for cancer. As used herein, the term “patient in need thereof” means any patient suffering from or predisposed to cancer. The term “patient in need thereof” means any patient in need of a low dose of a conventional cancer therapeutic agent. In addition, the term “patient in need thereof” means any patient in need of reducing the side effects of conventional therapeutic agents. Furthermore, the term “patient in need thereof” means any patient in need of improved tolerance to any conventional therapeutic agent for the treatment of cancer.

  The patient is typically an animal and more typically a mammal. As used herein, the term “mammal” means any animal classified as a human, domestic animal, zoo animal, sports animal or pet animal, such as a dog, horse, cat, cow, and the like. The patient may also be a human patient at risk of developing cancer or at risk of cancer recurrence.

  The methods and compositions of the invention may be used for the treatment or prevention of several cancers, including ovarian cancer, osteosarcoma, leukemia, lymphoma, small cell lung cancer, non-small cell lung cancer, Includes CNS cancer, breast cancer, colorectal cancer, kidney cancer, bladder cancer, breast cancer, epidermoid cancer, lung cancer, melanoma, prostate cancer, uterine cancer, soft tissue sarcoma, pancreatic cancer and rhabdomyosarcoma However, it is not limited to these.

  The compounds and formulations of the present invention can also be used for the treatment of colon cancer, ovarian cancer and osteosarcoma.

  The compounds and formulations of the invention can also be used for the treatment of osteosarcoma, colorectal cancer and pancreatic cancer.

  The following examples describe preferred embodiments of the invention. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention disclosed herein. The specification is to be regarded as illustrative only with examples, and the scope and spirit of the invention is indicated by the appended claims. In the following examples, all percentages are given on a weight basis unless otherwise indicated.

Example 1
This example is compared to 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereoisomer mixture and also 5 (R)-(2′-hydroxyethoxy) -20 ( FIG. 5 shows the unexpected and improved topoisomerase I inhibitory activity of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin when compared to the S) -camptothecin diastereoisomer.

<5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin, 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin, and 5 (RS)-(2 ' Preparation of -hydroxyethoxy) -20 (S) -camptothecin>:
A sample of 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin (75 grams) prepared as described in Example 26 of US Pat. No. 6,177,439 was transferred to n-butanol (about 600 mL) and refluxed for 2-3 hours. The temperature of the reaction mass is reduced to about 40-50 ° C. over about 1 hour, and the resulting solid material is filtered, washed with n-butanol (about 15-20 mL), and about 50-55 ° C. And 5 (R)-(2′-hydroxyethoxy) -20 (S) -solid 5 (S)-(2′-hydroxyethoxy) -20 (substantially free of camptothecin). S) -camptothecin was obtained. The product was further enriched by repeated refluxing in n-butanol to give 5 (S) -CPT substantially free of 5 (R) -CPT (generally 2 ~ 4 times; yield 25-35 grams).

  5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin was isolated from the mother liquor by dropwise addition of n-heptane followed by filtration using a 10μ Nutsche filter.

  5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin (a mixture of diastereomers) was obtained as described in Example 26 of US Pat. No. 6,177,439.

<Topoisomerase I test>:
Topoisomerase I introduces a temporary cut in DNA at specific sites. Detection of these temporary DNA breaks requires the use of a protein denaturant to trap the enzyme on the DNA in the cut intermediate complex. The resulting covalently bound DNA / topo I complex contains a nicked and open circular DNA that can be detected by agarose gel electrophoresis (using ethidium bromide). However, trapping nicked intermediates is relatively inefficient, and inhibitors such as the natural product camptothecin stabilize the intermediate, leading to an increase in nicked DNA products. . This forms the basis for mechanistic drug screening designed to allow detection of substances that affect topoisomerase by stabilizing the nicked intermediate complex.

  The TopoGEN® Topo I drug screening kit (Topogen, Inc., Port Orange, FL) is designed to allow researchers to rapidly identify new inhibitors of topoisomerase. The kit stabilizes nicked intermediates or allows detection of novel compounds that otherwise inhibit the catalytic activity of topoisomerase I.

<Assay kit used>: Topogen ^R drug screening kit Manufacturer: TOPOGEN, Cat No: 1018.
Each reaction mixture contains:
a. 1O x reaction buffer: 2 μL
b. TOPO I enzyme: 2 μL
c. pHOT I DNA: 1.2 μL (0.5 μg)
d. Water: 14.8 μL
Drug in DMSO: 1 μL
Total: 20 μL
<Protocol>
The above reaction mixture is incubated at 37 ° C. for 30 minutes. The reaction is stopped by adding 2 μL of 10% SDS, and the mixture is vortexed quickly (SDS is 37 ° C. as the chilled tube may close the nicked DNA again. Should be added in between). Add 10X dye, approximately 2.5 μL / tube, add an equal volume of a mixture of chloroform and isoamyl alcohol (24: 1) and centrifuge at 13000 rpm for 10 minutes. Samples are loaded on a 1% agarose gel and electrophoresed at 80 volts for 1 hour. The gel is observed with a UV light transmitter and a band density measurement evaluation is calculated.

<Calculation>:
The concentration of the superhelical and relaxed DNA bands was measured using a densitometer. Band intensities of the treatment group (with a single concentration of test drug) and the no drug group (ie, the control group) were recorded. The percentage of relaxed form of DNA as compared to superhelical form of DNA was calculated for all lanes including treatment and control groups.

The% inhibition of topoisomerase activity was calculated as follows:
= (100- (100 × (1 /% inhibition in control) ×% inhibition in treatment group)).

  Table 1 shows the results of these tests, and 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2'-) substantially free of each other. In vitro topoisomerase I activity of hydroxyethoxy) -20 (S) -camptothecin is shown relative to that of 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin.

Table 1: 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin, 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-(2 Topoisomerase I activity of '-hydroxyethoxy) -20 (S) -camptothecin Compound IC50 (μM)
5 (S)-(2'-Hydroxyethoxy) -20 (S) -camptothecin 1.65
5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin 22
5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin 12.5.

  This result shows that in the inhibition of topoisomerase I, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin. It is about 21 times as active as 5 and more than 12 times more active than 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin. Such differences in activity would not have been expected based on structural differences between diastereomers, especially since the importance of the E ring in enzyme activity is known.

Example 2
This example shows 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin compared to the activity of NCI-H460 (human small cell lung cancer) xenograft in nude mice. FIG. 3 shows the antitumor activity of S)-(2′-hydroxyethoxy) -20 (S) -camptothecin.

Samples of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin were as described in Example 1. Was provided.
<5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (S)-(2′-hydroxyethoxy) -20 (S) against NCI-H460 xenografts in nude mice -Protocol for comparative study of camptothecin>:
To do NCI-H460 xenograft studies, the NCI-H460 tumor pieces that had been weighed in to 60 mm ^3, were implanted in the space of the dorsal flank of female athymic mice (dorsal lateral flanks), begin to proliferate I let you. When tumors grew to ˜150-1000 mm ³ , animals were randomly divided into five groups before treatment began. Each gram of 5 (RS) -CPT was formulated to contain 102.65 mg of active compound, 801.62 mg of hydroxypropyl beta cyclodextran, 80.62 mg of anhydrous dextrose, and 13.33 mg of sodium carbonate. Each gram of 5 (S) -CPT was formulated to contain 105.57 mg of active compound, 800.99 mg of hydroxypropyl beta cyclodextran, 80.13 mg of anhydrous dextrose, and 13.34 mg of sodium carbonate. Each gram of placebo was formulated to contain 895.2 mg of hydroxypropyl beta cyclodextran, 89.52 mg of anhydrous dextrose, and 14.9 mg of sodium carbonate. Each formulation was dissolved in 2 mL of sterile water and administered via the oral route on a (dx5) 2 schedule. Tumor diameter was measured twice a week using calipers.

Assuming the tumor is elliptical, the tumor volume was calculated using the following formula:
V = (Dxd ² ) / 2
Where V (mm ³ ) is the tumor volume, D is the longest diameter in mm, and d is the shortest diameter in mm. The change in tumor volume (Δ) for each treatment group (T) and subject group (C) was calculated by subtracting the average tumor volume on the first day of treatment from the average tumor volume on a particular observation day. These values were used to calculate the growth percentage (% T / C) using the following formula:
% T / C = (ΔT / ΔC) × 100; where ΔT> 0,
Or% T / C = (ΔT / ΔTi) × 100; where ΔT <0
And Ti is the average tumor volume.

Tumor growth inhibition percentage (% TGI) was then calculated using the formula:% TGI = 100-% TC.

All mice with subcutaneous tumors measuring approximately 150-800 mm ³ were treated with test compounds by oral administration using the (dx5) 2 schedule. Tumor diameter was measured twice a week using calipers, and the tumor volume was calculated using the following equation, assuming that the tumor is elliptical:
V = (Dxd ² ) / 2
Where V (mm ³ ) is the tumor volume, D is the longest diameter in mm, and d is the shortest diameter in mm. The change in tumor volume (Δ) for each treatment group (T) and subject group (C) was calculated by subtracting the average tumor volume on the first day of treatment from the average tumor volume on a particular observation day. These values were used to calculate the growth percentage (% T / C) using the following formula:
% T / C = (ΔT / ΔC) × 100; where ΔT> 0,
Or% T / C = (ΔT / ΔTi) × 100; where ΔT <0
And Ti is the mean tumor volume at the start of treatment.

Tumor growth inhibition percentage (% TGI) was calculated using the formula:% TGI = 100-% T / C. Tumor regression is defined as partial when the tumor volume is reduced to less than 50% of the tumor volume at the start of treatment without dropping to less than 63 mm ³ . Complete regression is defined when the tumor volume falls below a measurable limit (<63 mm ³ ).

The percentage of body weight change compared to the body weight on the start date of each animal was calculated using the following formula:
Percentage change in body weight = [(weight on specific observation day−weight on start date) / weight on start date] × 100
Another parameter observed was mortality.

  Table 2 shows the results of the test. Here, tumor growth inhibition and mortality are shown for each of the two test compounds and the control.

Table 2: 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 () on tumor growth inhibition and mortality in nude mice bearing NCI-H460 (human small cell lung cancer) xenografts RS)-(2'-Hydroxyethoxy) -20 (S) -camptothecin Effect Compound Dose (mg / kg) Tumor growth inhibition% Mortality
5 (S)-(2'-hydroxyethoxy) 2 68 0/5
-20 (S) -camptothecin 4 76 0/5
5 (RS)-(2'-hydroxyethoxy) 2 60 0/5
-20 (S) -camptothecin 4 64 0/5.

  This test data shows that 5 (S)-(2'-hydroxyethoxy) -20 (S)-is more than the diastereoisomer mixture 5 (RS)-(2'-hydroxyethoxy) -20 (S) -camptothecin. It shows that camptothecin showed better in vivo activity against NCI-H460 (human small cell lung cancer) xenografts. As shown in Table 2, 5 (S)-(2'-hydroxyethoxy) -20 (S) compared to administration of 5 (RS)-(2'-hydroxyethoxy) -20 (S) -camptothecin -Administration of camptothecin led to unpredictable superior tumor growth inhibition at the same dose without increasing mortality (68% vs 60% at 2 mg / kg and 76 at 4 mg / kg % Vs 64%).

Example 3
This example shows 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin vs. 5 (R)-(2'- in inhibiting cell growth in vitro in the sulforhodamine B (SRB) test. The efficacy of hydroxyethoxy) -20 (S) -camptothecin is shown.

  Samples of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin were as described in Example 1. Was provided.

<Protocol for in vitro cell proliferation test>
Cell proliferation is assessed by the sulforhodamine B (SRB) test, where the amount of dye bound to the cells after staining gives a measure of cell proliferation. See JNCI, vol 83, No. 11, June 5, 1991, incorporated herein by reference.

Briefly, cells (34 human cancer cell lines represented by bladder cancer, breast cancer, CNS cancer, colon cancer, epidermoid cancer, lung cancer, ovarian cancer, melanoma, prostate cancer, kidney cancer, and uterine cancer) The cells were seeded in 96-well cell culture plates at a concentration of 10,000 cells per well and incubated at 37 ° C. in a CO ₂ incubator. After 24 hours, the medium was treated with different concentrations of test compound dissolved in DMSO to a final concentration of 0.05% and exposed for 48 hours. The cells were fixed by adding ice-cold 50% trichloroacetic acid (TCA) and incubating at 4 ° C. for 1 hour. The plates were washed with distilled water, air dried and stained with SRB solution (0.4% wt / vol in 1% acetic acid) for 10 minutes at room temperature. Unbound SRB was removed by extensive washing with 1% acetic acid and the plates were air dried. Bound SRB staining was solubilized with 10 mM Tris buffer and the optical density was read at a single wavelength of 515 nm in a spectrophotometer plate reader. At the time of drug addition, another reference plate for cell growth at zero time (when drug was added) was also measured as described above. From the optical density, the growth percentage was calculated using the following formula:
If T is greater than or equal to T ₀ ,
Proliferation percentage = 100 × [(T−T _o ) / (C−To)]
If T is less than T ₀ ,
Proliferation percentage = 100 × [(T−T ₀ ) / T ₀ )]
Where T is the optical density of the test, C is the optical density of the control, and T ₀ is the optical density at zero.

A dose response curve was generated from the growth percentage and GI ₅₀ values were interpolated from the growth curve. Table 3 shows the results.

Table 3: 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin vs 5
GI ₅₀ value of (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin Compound GI ₅₀ value
5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin 5.0
5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin 14.6.

  The results of this test show that 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin diastereomer has a 5 (R)-(2′-hydroxy It was almost 3 times more active than (ethoxy) -20 (S) -camptothecin.

Example 4
This example demonstrates the efficacy of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin in several osteosarcoma tumor models.

  Samples of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin were as described in Example 1. Was provided.

  This test was performed as described in Cancer Res., Oct. 15:64 (2) 7491-9 (2004), and Clin. Cancer Res., Nov. 15: 9 (15): 5442-53 (2003). Made using.

All mice with subcutaneous ("sc") tumors measuring approximately 0.2-1 cm in diameter were treated with test compounds by oral administration using the [(dx5) 2] 3 schedule. Tumor diameter was measured daily for 7 days using calipers, and the tumor volume was calculated using the formula [式 / 6) xd ³ ], assuming the tumor was spherical. Here, d is an average diameter. Tumor response to test compounds was defined as follows: For individual tumors, partial regression (PR) was defined as having a volume reduction> 50% but having measurable tumors at all time points It was done. Complete regression (CR) was defined as measurable disappearance of the tumor mass at several time points within 12 weeks after the start of treatment. Persistent CR is defined as no tumor regrowth within the 12 week study time frame. This time point was chosen because all studies last at least 12 weeks. Mice that died before the end of the 12-week laboratory and before achieving a response were considered to have had no tumor response. The results (28 mg / kg dose) are presented in Table 4.

Table 4: Effects of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin vs. 5 (RS)-(2'-hydroxyethoxy) -20 (S) -camptothecin in a mouse tumor regression model Xenograft 5 (S)-(2'-hydroxyethoxy) 5 (RS)-(2'-hydroxyethoxy)
-20 (S) -camptothecin -20 (S) -camptothecin
Osteosarcoma 29 6+ 5+
Osteosarcoma 17 6+ 4+
Osteosarcoma 2 6+ 5+
Osteosarcoma 32 6+ 3+
6+; complete regression maintained
5+; complete regression
4+; partial challenge
3+; stable disease As shown by the data in Table 4, 5 (S) − as shown by the fact that sustained complete regression (6+) was achieved in all four xenografts. (2'-Hydroxyethoxy) -20 (S) -camptothecin administration was an unexpected and superior result compared to 5 (RS)-(2'-hydroxyethoxy) -20 (S) -camptothecin administration Led.
The data given in Examples 3 and 4 show that 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin has superior activity / capacity that was not expected in some test models. It is shown that. Furthermore, 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is substantially more potent than 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin On the other hand, unexpectedly, this increase in capacity is not accompanied by a corresponding increase in toxicity.

Example 5
This example illustrates the human myelotoxicity of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin vs. 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin Is shown.

  Samples of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin are described in Example 1. As provided.

<Protocol for human bone marrow test>
For this study, Methocult® GF containing methylcellulose, fetal bovine serum, bovine serum albumin, 2-mercaptoethanol, L-glutamine, rh stem cell factor, rhGM-CSF and rhIL-3 in Iscove's MDM ( Cat No: H4534, Poietics, Biowhittakar, USA) medium. Human bone marrow monocytes (Cat No. 2M-125C, Poietics, Biowhittakar, USA) were mixed with Methocult® GF and the cell density was adjusted to 3 × 10 ⁵ cells / mL. 500 μL aliquots were made from this preparation, and 2.5 μL of 2O × drug solution or carrier was added to each aliquot and mixed well. 100 μL of clonogenic medium was plated into each well and the plate was allowed to gel at 4 ° C. for 15 minutes. Plates were incubated for 14 days at 37 ° C. in a fully humidified 5% CO ₂ atmosphere in an incubator. CFU-GM colonies were counted under an inverted microscope as aggregates of 50 cells or more. The percentage of colony inhibition was calculated using the following formula:
100-[(number of colonies in drug-treated well / number of colonies in control well) × 100]
The in vitro ability of the two diastereomers against cancer cell lines was compared to their in vitro toxicity in healthy cells. Table 5 presents the results of a bone marrow toxicity comparison study in human cells.

Table 5: 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin for human bone marrow cells in vitro GI ₉₀
Compound GI ₉₀
5 (S)-(2'-Hydroxyethoxy) -20 (S) -camptothecin 0.69
5 (R)-(2'-Hydroxyethoxy) -20 (S) -camptothecin 0.89
Referring to the data shown in Table 3 and Table 5, 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin, contrary to expectation, was found to be a human cancer cell line of 34 species (bladder cancer, Breast cancer, CNS cancer, colon cancer, epidermoid cancer, lung cancer, ovarian cancer, melanoma, prostate cancer, kidney cancer, and uterine cancer) for 5 (R)-(2'-hydroxyethoxy) -20 ( Although it is almost 3 times more potent than S) -camptothecin, it can be seen that the toxicity of both diastereomers is comparable. In fact, as shown in Table 6, if safety margin is evaluated as the ratio of GI ₉₀ for human cytotoxicity to GI ₅₀ for anticancer activity, 5 (S)- (2'-hydroxyethoxy) -20 (S) -camptothecin is a pharmaceutical compound for the treatment of cancer as 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-( It is clearly superior to 2'-hydroxyethoxy) -20 (S) -camptothecin. In other words, the 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin compound has increased efficacy in treating cancer compared to the R-diastereomers and diastereomeric mixtures. is doing. In fact, the amount of 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin present in 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin given to the patient It is unexpectedly important to minimize.

Table 6: Anticancer activity GI _{50 for} 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin Ratio of human cytotoxic GI ₉₀ to compound safety margin (GI ₉₀ / GI ₅₀ )
5 (S)-(2'-hydroxyethoxy) 0.14
-20 (S) -camptothecin
5 (R)-(2'-hydroxyethoxy) 0.06
-20 (S) -camptothecin.

Example 6
This example shows 5 (R)-(2'-hydroxyethoxy) -20 (S) for the bioavailability of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin in rats and mice. -Shows the effect of the presence of camptothecin.

  5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin ("5 (S) -CPT") and 5 (RS)-(2'-hydroxyethoxy) -20 (S) -camptothecin ( A sample of “5 (RS) -CPT”) was provided as described in Example 3.

<Bioavailability in male Wistar rats>
To assess oral pharmacokinetics, 5 (S) -CPT (2.5 mg / kg) and 5 (RS) -CPT (5 mg / kg, 2.5 mg / kg of 5 (S) -CPT in the mixture Were administered to male Wistar rats.

  Male Wistar rats aged 6-8 weeks and weighing 205-218 g were divided into groups of 4 rats. Oral pharmacokinetic studies were performed under overnight fasting conditions, and intravenous pharmacokinetics were performed under dietary conditions. The test drug was administered as a solution by oral gavage or by lateral tail vein injection. Approximately 250 microliters of low density blood samples were collected from the retro-orbital plexus into a microcentrifuge tube containing 25 microliters of EDTA at the time of design. Plasma was separated by centrifuging the blood at 12,800 rpm for 2 minutes and refrigerated until analysis.

  Samples were tested for the presence of the test drug as follows. An aliquot of 100 μL of plasma (stored at 8 ° C.) was precipitated with 400 μL of cold methanol to evaluate the total (lactone + carbonate). After mixing for 2 minutes and centrifuging for 4 minutes at 12,800 rpm, the clear supernatant was separated into a 300 μL automatic sampler bottle and 20 μL of this mixture was injected onto an analytical column for HPLC analysis. The concentration of the test drug was calculated from the plotted linearity by spiking a known concentration of the test drug into blank rat plasma. The pharmacokinetics of the study drug was calculated using non-compartmental analysis. The results of this study are presented in Table 7.

Table 7: Oral pharmacokinetic parameters of 5 (S) -CPT in male Wistar rats Compound Dose AUC (ot) μM ^* h
5 (S) -CPT 5 mg / kg 5.76
5 (RS) -CPT 5 mg / kg 5.08
(2.5 mg / kg 5 (S) -CPT
+ 2.5 mg / kg 5 (R) -CPT)
2.5 mg / kg 1.21 in 5 (RS) -CPT
5 (S) -CPT contribution.

<Bioavailability in Swiss albino mice>
Oral pharmacokinetics of 5 (S) -CPT (2.5 mg / kg) and 5 (RS) -CPT (5 mg / kg, containing 2.5 mg / kg of 5 (S) -CPT in the mixture) Was administered to Swiss albino mice.
Swiss albino mice 3 to 6 weeks old and weighing 28 to 34 g were used for the study. Twelve mice were used per study. Oral pharmacokinetic studies were performed under overnight fasting conditions, and intravenous pharmacokinetics were performed under dietary conditions. The test drug was administered as a solution by oral gavage or by lateral tail vein injection. Approximately 250 microliters of sparse blood samples were collected from the retro-orbital plexus into a microcentrifuge tube containing 25 microliters of EDTA at the time of design. Plasma was separated by centrifuging the blood at 12,800 rpm for 2 minutes and refrigerated until analysis.

  Samples were tested for the presence of the test drug as follows. An aliquot of 100 μL of plasma (stored at 8 ° C.) was precipitated with 400 μL of cold methanol to evaluate the total (lactone + carbonate). After mixing for 2 minutes and centrifuging for 4 minutes at 12,800 rpm, the clear supernatant was separated into a 300 μL automatic sampler bottle and 20 μL of this mixture was injected onto an analytical column for HPLC analysis. The concentration of the test drug was calculated from the plotted linearity by spiking a known concentration of the test drug into blank rat plasma. The pharmacokinetics of the study drug was calculated using non-compartmental analysis. The results of this study are presented in Table 8.

Table 8: Oral pharmacokinetic parameters of 5 (S) -CPT in Swiss albino mice Compound Dose AUC (ot) μM ^* h
5 (S) -CPT 5 mg / kg 5.18
5 (RS) -CPT 5 mg / kg 5.20
(2.5 mg / kg 5 (S) -CPT
+ 2.5 mg / kg 5 (R) -CPT)
2.5 mg / kg 1.10 in 5 (RS) -CPT
5 (S) -CPT contribution.

  Referring to Tables 7 and 8, “5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin contribution” refers to the S-diastereomer in the RS diastereomeric mixture. Is the area under the curve ("AUC"). As can be seen from Tables 7 and 8, the presence of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin is unexpectedly desirable for the 5 (S) -CPT diastereomeric organism. Reduce usability. In addition, such a diminished bioavailability of desirable diastereomers would be observed in human patients. Based on the above, the inventors have recognized that it is desirable to minimize the amount of R diastereomer in 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin.

Example 7
This example shows 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin vs. 5 (R)-for Saos-2 cells overexpressing BCRP variants and breast cancer resistance protein (BCRP). The efficacy of (2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin is shown.

5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin, 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-(2'-hydroxy A sample of ethoxy) -20 (S) -camptothecin was provided as described in Example 1.
<Protocol for Breast Cancer Resistance Protein (BCRP) Test>
5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (R)-(2'- against Saos-2 overexpressing functional BCRP # 4 and non-functional BCRP mut # 10 The anticancer effect of hydroxyethoxy) -20 (S) -camptothecin was evaluated against racemic 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin. The human osteosarcoma cell line, Saos-2, was obtained from ATCC (American Type Culture Collection, Cat # HTB-85, Manassas, VA), 10% fetal bovine serum, 1% penicillin / streptomycin, and 2 mM. Maintained in DMEM containing glutamine. Saos-2 cells were transfected with BCRP # 4 to overexpress functional BCRP, or BCRP # 10 was transfected to overexpress nonfunctional BCRP transporter. The cells were seeded overnight at a density of 1000 cells per well in 96-well plates in 0.1 mL medium. The next morning, the medium was gently aspirated and a serial dilution of the compound to be tested was added. Cells were incubated at 37 ° C. in a 5% CO ₂ incubator. After 6 days of exposure to the test drug, 10 μL of Alamar blue was aseptically added to each well and the plate was returned to the incubator for 6 hours. The amount of fluorescent dye produced was measured on a Cytofluor 2300 (Millipore, Bedford, Mass.) Using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The relative fluorescence units obtained were used to calculate the percentage growth at each concentration relative to the untreated control value. From this growth percentage value, an IC ₅₀ (inhibitory concentration required to inhibit 50% cell growth compared to control cell growth) value was derived. The resulting IC ₅₀ values are presented in Table 9.

Table 9: Saos-2 overexpressing BCRP variants and breast cancer resistance protein (BCRP)
5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin, 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-(2 IC ₅₀ value of '-hydroxyethoxy) -20 (S) -camptothecin Drug IC ₅₀ (nM)
BCRP mut # 10 BCRP # 4
5 (RS) -CPT (2'-hydroxyethoxy) 387 1256
-20 (S) -camptothecin
5 (S) -CPT (2'-hydroxyethoxy) 213 788
-20 (S) -camptothecin
5 (R) -CPT (2'-hydroxyethoxy) 1299> 2000.

-20 (S) -camptothecin As shown in Table 9, 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is a BCRP mutant as well as its counterpart to Saos-2 overexpressing BCRP. It is superior to 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin and 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin in terms of cytotoxic activity. These results indicate that the cytotoxicity ranking for both BCRP mut # 10 and BCRP # 4 is 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin> 5 (RS)-(2 '-Hydroxyethoxy) -20 (S) -camptothecin> 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin. 5 (S)-(2′-Hydroxyethoxy) -20 (S) -camptothecin is better than BCRP mut # 10 and BCRP # than 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin. It was ˜6-fold and> 2.5-fold cytotoxicity against Saos-2 cells overexpressing 4, respectively.

  Although the invention has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Accordingly, it will be appreciated that many variations may be made to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the claims, and that other configurations may be devised. Should.

  Many publications cited in the specification (including all papers, publications, patents, patent applications, presentations, texts, reports, handwritten manuscripts, brochures, books, Internet postings, journal articles, periodicals, etc. (Which are not limited to these) are incorporated herein in their entirety. The discussion of cited references herein is merely intended to summarize the assertions made by their authors and does not admit that any cited reference constitutes prior art. Applicant reserves the right to refute the accuracy and appropriateness of the cited documents.

  In view of the above, it will be seen that the several advantages of the invention are achieved and that several advantageous results are obtained.

  Those skilled in the art will be able to make various changes in the above methods and compositions without departing from the scope of the present invention, so all matters contained in the above description and the accompanying drawings are shown. It is intended that the matter should be construed as illustrative and not in a limiting sense. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part.

Claims (29)

  5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin substantially free of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin.   5. 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin according to claim 1, which is optically substantially pure.   5. 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin according to claim 1 having a chemical purity of greater than 98%.   The 5 (S)-(2'-hydroxyethoxy) of claim 1 wherein 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin is present in an amount of about 1.5% w / w. -20 (S) -camptothecin.   The 5 (S)-(2'-hydroxyethoxy) of claim 1 wherein 5 (R)-(2'-hydroxyethoxy) -20 (S) -camptothecin is present in an amount less than about 1% w / w. ) -20 (S) -camptothecin.   The 5 (S)-(2'-hydroxyethoxy) -5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is present in an amount of less than about 0.5% w / w. ) -20 (S) -camptothecin.   The 5 (S)-(2'-hydroxyethoxy) -20 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is present in an amount less than about 0.1% w / w. ) -20 (S) -camptothecin.   A camptothecin dimer impurity having a chemical structure of Formula V is present in 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin in an amount less than about 1.5% w / w. 5. 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin according to 1.   A camptothecin dimer impurity having a chemical structure of Formula V is present in 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin in an amount less than about 1% w / w. 5. 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin according to 1.   The camptothecin dimer impurity having the chemical structure of Formula V is present in 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin in an amount less than about 0.1% w / w. 5. 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin according to 1.   A method of inhibiting the activity of topoisomerase I in a cell, said method being substantially free of 5 (R)-(2′-hydroxyethoxy) -20 (S) -camptothecin with respect to said cell Administering an inhibitory effective amount of 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin.   12. The method of claim 11, wherein the 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is administered to the cells in vitro.   12. The method of claim 11, wherein the 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is administered to the cells in vivo.   A pharmaceutical composition comprising 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin according to claim 1 and a pharmaceutically acceptable carrier.   15. The pharmaceutical composition of claim 14, wherein a single dose of the composition for a human adult is from about 5 mg to about 25 mg of 5 (S)-(2′-hydroxyethoxy) -20. A composition comprising (S) -camptothecin.   Use of 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin for the manufacture of a pharmaceutical composition comprising the 5 (S)-(2 Mixing '-hydroxyethoxy) -20 (S) -camptothecin with a pharmaceutically acceptable carrier. A process for producing 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin according to claim 1, comprising the steps of:
a) mixing 5 (RS)-(2′-hydroxyethoxy) -20 (S) -camptothecin with a solvent selected from n-butanol or tetrahydrofuran;
b) refluxing the mixture for about 1 to about 4 hours;
c) cooling the mixture to a temperature of about 40 ° C. to about 50 ° C .;
d) A method comprising separating solid 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin from the mixture. 18. The method of claim 17, further comprising:
e) washing the solid 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin with a solvent selected from n-butanol or tetrahydrofuran;
d) The solid 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin is dried to give substantially 5 (R)-(2′-hydroxyethoxy) -20 (S) — A process comprising obtaining 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin free of camptothecin.   A method of using 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin for treating cancer in a patient comprising: an effective amount of claim for said patient 4. A method comprising administering 5 (S)-(2′-hydroxyethoxy) -20 (S) -camptothecin according to 1.   21. The method of claim 19, wherein the patient is a mammal.   20. The method of claim 19, wherein the patient is a person.   21. The method of claim 19, wherein the 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is administered orally to the patient.   20. The method of claim 19, wherein the cancer is ovarian cancer, osteosarcoma, leukemia, lymphoma, small cell lung cancer, non-small cell lung cancer, central nervous system (CNS) cancer, breast cancer, colorectal cancer, A method which is selected from the group consisting of kidney cancer, bladder cancer, breast cancer, epidermoid cancer, lung cancer, melanoma, prostate cancer, uterine cancer, soft tissue sarcoma, pancreatic cancer and rhabdomyosarcoma.   24. The method of claim 23, wherein the cancer is selected from the group consisting of colon cancer, ovarian cancer, and osteosarcoma.   24. The method of claim 23, wherein the cancer is selected from the group consisting of osteosarcoma, colorectal cancer and pancreatic cancer.   24. The method of claim 23, wherein the cancer comprises bladder cancer, breast cancer, CNS cancer, colorectal cancer, epidermoid cancer, lung cancer, ovarian cancer, melanoma, prostate cancer, kidney cancer, and uterine cancer. A method that is selected from:   20. The method of claim 19, wherein the 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is administered to the patient in an amount of about 0.1 to about 10 mg / kg / day. How to be.   21. The method of claim 19, wherein the 5 (S)-(2'-hydroxyethoxy) -20 (S) -camptothecin is administered to the patient in an amount of about 0.1 to about 5 mg / kg / day. How to be.   21. The method of claim 19, wherein the patient is in need of treatment for cancer.

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