DT-DIAPHORASE-ACTIVE AGENTS FOR TREATMENT OF PEDIATRIC

TUMORS

BACKGROUND

[0001] DT-Diaphorase (DTD) was first isolated in 1958 and has been referred to by a variety of names including NAD(P)H: quinone oxidoreductase (EC 1.6. 99.2)(NQO1), vitamin K reductase, phylloquinone reductase, menadione reductase and nicotinamide menaione oxidoreductase.

[0002] DTD is a flavoprotein which exists as a dimer. Both subunits are of equal size, have MW of 32000 Dalton and have 2 FAD groups. DTD is an obligatory two electron reductase enzyme (in contrast to the one electron reductase enzymes such as cytochrome b reductase, cytochrome P450 reductase and xanthine dehydrogenase) and utilises co- factors NADH and NADPH equally well as the electron donor.

[0003] DTD performs a number of functions including Phase II detoxification, a detoxifying step that bypasses the formation of free radicals and protects tissue against mutagens, carcinogens and cytotoxics. DTD also metabolisms quinones (e.g. originating from diet or the environment). In particular it can reductively activate cytotoxic antitumour quinones. Furthermore, DTD functions as a vitamin K reductase involved in hepatic post- translational modification of vitamin K. DTD is distributed throughout the body with higher levels in the liver, kidney and gastrointestinal tract. There are four different isoforms of DTD. The best characterized is NQO 1 and the gene for this isoform is located on chromosome 16. It is 274 residues long and has an ARE (antioxidant response element), API site, XRE, CAT, TATA box and NFkB binding site. Binding to ARE mediates signal transduction (Faig et al. PNAS 28, 3177-82, 2000).

[0004] Elevated levels of DTD can be found in certain tumour types, compared to normal tissue (Schlager et al., Int. J. Cancer 45, 403-409, 1990).

[0005] US6156744, incorporated herein in its entirety by reference, proposes the use of 2,5-diaziridinyl-3-hydroxymethyl-6-methyl 1,4- benzoquinone (RHl) and certain esters of RHl for the treatment of lung cancer, NSCLC, liver, breast, colon, CNS, stomach, bladder and skin cancer. WO 2005/107743, also incorporated herein in its entirety by reference, proposes dosage levels of one particular compound, 2,5-diaziridinyl-3-(hydroxymethyl)-6- methyl-l,4-benzoquinone (RHl), which are particularly effective at certain dosage level.

[0006] Children are subject to a number of cancers, which are often resistant to normal chemotherapeutic agents. The following list includes the most common types of cancer that occur in children.

[0007] Ewing's sarcoma: The Ewing's family of tumors includes cancerous tumors affecting soft tissue or bone. This rare disease most often occurs in children or young adults. The most common type of Ewing's tumor, accounting for more than 80 percent of Ewing's cases, grows in or near bones, most commonly the large bones in the leg (femur and tibia) and the upper arm bone (humerus). Ewing's sarcoma may also occur in other bones or tissue. [0008] Neuroblastoma: Neuroblastoma begins in nerve tissues in the abdomen, pelvis, neck, or chest. The tumors often grow rapidly and, when they spread into other organs or parts of the body, are difficult to treat successfully. Neuroblastoma is one of the more common forms of cancer in infants and children. Physicians diagnose approximately 525 cases in the United States each year. Neuroblastoma occurs in about 10 per one million children. Experts do not know its cause, and many cases may actually be present at birth. The disease is usually found before the child reaches age 2.

[0009] Osteosarcoma: Around 2,000 cases are diagnosed in the United States each year. The most common type, accounting for 35 percent of bone cancer cases, is osteosarcoma, a tumor that develops from cells that form bone. The bones most often affected are the large bones in the lower extremity (femur and tibia) and the upper arm bone (humerus), although osteosarcoma may occur in any bone. If the osteosarcoma tumor is found in one location and has not spread to other parts of the body, the cure rate is 65 to 75 percent. Osteosarcoma is most prevalent in people between the ages of 10 and 30. [0010] Other pediatric tumors include leukemia, Rhabdomyosarcoma, soft tissue sarcoma, and Wilm's tumors.

[0011] While the compound RHl has been found effective for certain cancers, it is desirable to identify other cancers which may be responsive to this compound. Despite dramatic improvements in survival from childhood cancer there remain many tumor types in which drug resistance is a major problem. There is thus an urgent need for access to effective agents for this group of patients.

[0012] The present invention is directed toward overcoming one or more of the problems discussed above.

DETAILED DESCRIPTION

[0013] The present inventors have found that certain diaziridinylbenzoquinone compounds are suitable for treatment of a wide range of cancerous conditions, and that such compounds are particularly effective when administered to a patient at certain dosage levels, optionally as part of a predetermined dosage regime, and/or using certain modes of administration.

[0014] 2,5 Diaziridinylbenzoquinone compounds of the invention include the compound 2,5-diaziridinyl-3-hydroxymethyl-6-methyl-l,4 benzoquinone (herein called RHl) and its esters. The structure of RHl is given by Formula I:

[0015]

[0016] Reference herein to RHl or compounds of Formula I includes the salts thereof, in particular pharmaceutically acceptable salts thereof.

[0017] The present inventors have found that some cell lines derived from pediatric tumors express DTD. Previous work focused on observed over-expression of DTD in tumours and the efficient activation of compounds of the present invention by DTD means that compound activation occurs preferentially within the tumour. Tumours with high DTD levels preferably exhibit correspondingly higher levels of DNA cross-linking when treated with compounds of the present invention, particularly RHl.

[0018] RHl is a bioreductively activated drug that has been found to be an excellent substrate for DTD. DTD reduces RHl to a hydroquinone producing a powerful cross-linking agent. Compounds of the present invention, including RHl, can be thought of as pro-drugs, in the sense that they are metabolized by DTD to convert them into their active form.

[0019] It has been found that compounds of the present invention, in particular RHl, can cross-link DNA. Preferably significant cross-linking (e.g. in the range 40-95%) is caused, which may lead to accumulative DNA damage. For example, compounds or compositions of the present invention, e.g. containing RHl, may be administered to cause at least 10% cross- linking in DNA, more preferably at least 20% cross-linking, even more preferably at least 30% cross-linking, still more preferably at least 40% cross- linking and most preferably at least 50% cross-linking: For example, RHl has been found to cause up to 30% cross-linking in DNA of peripheral blood lymphocytes.

[0020] The present inventors have found that for the pediatric tumors neuroblastoma,

Ewing's sarcoma and osteosarcoma, cell lines derived from those particular tumors express differential amount of DTD. See Example 1. Surprisingly, the present inventors have found that there was no correlation between clonogenic survival and DTD expression in RHl treated cells from cell lines from these particular tumors, and IC50 ranged from 1.5 to 7.5 nM, even in cell lines relatively resistant to cisplatin and doxorubicin. [0021] It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of RHl, preferably a pharmaceutically- acceptable salt. It is preferred that the salt is water soluble. Examples of pharmaceutically acceptable salts are discussed in Berge et al, 1977, 2 0 "Pharmaceutically Acceptable Salts," J. Pharm. ScL, Vol. 66, pp. 1- 19. Accordingly, aspects of the invention may include any known pharmaceutically acceptable salt of RHl. Accordingly, compounds, compositions, uses and methods of the invention which refer to compounds of the present invention, in particular 2, 5- diaziridinyl- 3-hydroxymethyl-6-methyl-l,4 3 0 benzoquinone (RHl), may include salts, preferably pharmaceutically acceptable salts of the compounds, in particular of 2, 5-diaziridinyl-3- hydroxymethyl-6-methyl-l, 4 benzoquinone (RHl).

[0022] In a further aspect the present invention provides pharmaceutical compositions comprising diaziridinylbenzoquinone compounds of formula I or salts thereof for the methods of the invention, in particular comprising 2,5-diaziridinyl-3 hydroxymethyl- 6-methyl-l,4-benzoquinone (RHl). Preferably, such compositions comprise one or more pharmaceutically acceptable carriers, adjuvants or diluents. The compounds can be formulated in any pharmaceutically acceptable formulation. Such formulations may include liquids, powders, creams, emulsions, pills, troches, suppositories, suspensions, solutions, and the like. Other excipients can also be added Io and are readily identified by those skilled in the art. Preferably, the compounds are soluble in aqueous solutions, are stable, and can be

prepared in gram quantities. For example, formulations may be in tablet form, or suitable for injection, e.g. combined with an appropriate fluid carrier.

[0023] Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes, including but not limited to, topical, parenteral, intravenous, intramuscular, intratumoral, intrathecal, intraocular, subcutaneous, transdermal, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form, for example as an injectable composition or in tablet form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body, typically combined with an appropriate fluid carrier. Preferred routes of administration of the compound or composition may include one or more selected from topical, parenteral, intravenous, intramuscular, intratumoral, intrathecal, intraocular, subcutaneous, transdermal, oral and nasal. A particular formulation of the compound, e. g. RHl, may be selected to correspond to the route of administration that is to be used.

[0024] Aspects of the present invention relate to the treatment of cancerous conditions. As such, a method of treating a cancerous condition in a patient comprising administering to said patient a therapeutically- effective amount of a compound of the present invention is provided. Thus, methods of treating patients, including human patients, having cancer are provided.

[0025] Treatment may be by administration of 2,5-diaziridinyl-3-hydroxymethyl-6- methyl-1, 4- benzoquinone (RHl) or compositions containing 2,5- diaziridinyl-3- hydroxymethyl-6- methyl-l,4-benzoquinone (RHl). Suitably, the compound or pharmaceutically acceptable salt thereof is part of a composition and it is the composition that is administered.

[0026] Therapeutically effective amounts of the compounds can be any amount or dose sufficient to bring about the desired therapeutic effect (e.g. killing of tumour cells) and may I depend, in part, on factors such as the condition, type and location of the cancerous condition being treated, as well as the size and condition of the patient. The dosages can be given as a single dose, or as several doses, for example, divided over the course of several weeks. The dosages may be administered as part of a predetermined program of treatment. [0027] A therapeutically effective amount may be one that produces at a given time after administration a blood, plasma or serum concentration of RHl in the patient which is in the range 30 to 12OnM, more preferably in the range 50 to 9OnM. A serum sample may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood

cells. A therapeutically effective amount may be one that is in the range 40 ug/m /day to 350 ug/m ² /day.

[0028] A predetermined time interval may be provided between dosages. For example, a dosing schedule may be provided in which a selected dose is administered daily (i.e. at 24 hour intervals) for a number of days (e.g. any of 1, 2, 3, 4, 5, 6 or 7 days) and then a further time interval (e.g. 1, 2, 3, 4, 5, 6 or 7 days) is provided during which no drug is administered (i.e. a 'drug holiday'). Each period of drug administrations followed by 'drug holiday' may comprise one cycle of treatment. A dosing routine may be provided having any number of cycles, as desired to achieve treatment. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles may be provided. The patient may then be removed from treatment, which of course, may be re-commenced if further treatment is considered necessary. [0029] The cancerous condition may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumor. The cancerous condition may be a cancer and may be a benign or malignant cancer and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue.

[0030] Examples of such cancer types include pediatric tumor, such as, for example, neuroblastoma, osteosarcoma, and Ewing's sarcoma. Cancerous conditions selected for treatment may be those that have proven to be resistant to (are refractory to) treatment with conventional chemotherapy or radiotherapy.

[0031] Methods of treating patients, including human patients, having a cancerous condition are provided. Treatment may be by administration (e.g. by injection, orally, eta) of compounds or compositions according to the present invention, preferably I of 2,5- diaziridinyl-3-hydroxymethyl-6- methyl- 1,4-benzoquinone (RHl) or compositions containing 2,5-diaziridinyl- 3 hydroxymethyl-6-methyl- 1,4-benzoquinone (RHl). [0032] The patient to be treated may be any animal or human. The patient may be a non-human mammal, but is more preferably a human patient. The patient may be male or female.

[0033] In one embodiment, RHl is administered together with an effective amount of cisplatin. Cisplatin, cisplatinum or cis-diamminedichloroplatinum(II) (CDDP) is a platinum- based chemotherapy drug used to treat various types of cancers, including sarcomas, some carcinomas (e.g. small cell lung cancer, and ovarian cancer), lymphomas and germ cell tumors. Upon administration, a chloride ligand undergo slow displacement with water (an

aqua ligand) molecules, in a process termed aquation. The aqua ligand in the resulting [PtCl(H ₂ O)(NH ₃ ) ₂ ] ⁺ is easily displaced, allowing cisplatin to coordinate a basic site in DNA. Subsequently, the platinum cross-links two bases via displacement of the other chloride ligand. Cisplatin crosslinks DNA in several different ways, interfering with cell division by mitosis. The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible. Most notable among the DNA changes are the 1,2- intrastrand cross-links with purine bases. These include 1,2-intrastrand d(GpG) adducts which form nearly 90% of the adducts and the less common 1,2-intrastrand d(ApG) adducts. 1,3-intrastrand d(GpXpG) adducts occur but are readily excised by the nucleotide excision repair (NER) . Other adducts include inter-strand crosslinks and nonfunctional adducts that have been postulated to contribute to cisplatin's activity.

[0034] Doxorubicin (trade name Adriamycin) ((8S,10S)-10-(4-amino-5-hydroxy-6- methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,l l-trihydroxy-8-(2-hydroxyacetyl)-l-methoxy- 7,8,9,10 tetrahydrotetracene-5,12-dione) or hydroxyldaunorubicin is a DNA-interacting drug widely used in chemotherapy. It is an anthracycline antibiotic and structurally closely related to daunomycin, and also intercalates DNA. It is commonly used in the treatment of a wide range of cancers. The exact mechanism of action of doxorubicin is complex and still somewhat unclear, though it is thought to interact with DNA by intercalation. Doxorubicin is known to interact with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which unwinds DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication.

[0035] In some methods of the invention, both RHl and cisplatin or RHl and doxorubicin are administered to mammals (e.g., humans, male or female) using respective conventional methods. Administration of each composition referenced herein can be in a dosage form and schedule in accordance with current protocols, recommendations, or schedules known in the art for that composition and/or compound. In this embodiment, the administration of the RHl and cisplatin/doxorubicin will be in accordance with protocols and/or dosing regimes specific to each, but will occur in a manner that administration of RHl and cisplatin/doxorubicin are at least partially overlapping in a specific mammal during a specific treatment regimen. In one embodiment, the administration of RHl and cisplatin/doxorubicin is substantially overlapping during a treatment regimen. In one

embodiment, the treatment regimens for RHl and cisplatin/doxorubicin will overlap sufficiently in order for the beneficial effects as noted herein to occur. [0036] While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.

EXAMPLES

[0037] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1:

[0038] DT-Diaphorase expression in individual cell lines corresponding to neuroblastoma, osteosarcoma, and Ewing's sarcoma.

[0039] Individual cell lines SH-EPl, SY5Y, LA1-5S, LAl-FFN (corresponding to neuroblastoma), 79 IT and U2OS (corresponding to osteosarcoma) and A673 and RDES (corresponding to Ewing's sarcoma) were compared for DTD protein levels. See Figure 1. The data shows that for neuroblastoma cell lines tested, SH-EP 1 appeared to have greater expression of DTD relative to the control, and such level was higher than SY5Y, LA 1-5 S, LAl-FFN, which appeared to express significantly less DTD than control. For osteosarcoma cell lines tested, 79 IT appeared to have greater expression of DTD relative to the control, and U2OS also had measurable expression, but less than 79 IT and approximately equivalent to control. For Ewing's sarcoma cell lines tested, A673 appeared to have approximately equal expression to the control, and RDES appeared to have less expression than control. Positive expressers of DTD were deemed to include SH-EPl, LA 1-5 S, 79 IT, and A673.

Example 2:

[0040] RHl's toxicity against pediatric tumor cell lines

[0041] Figure 2 shows clonogenic survival of all cell lines noted in Example 1 following one hour treatment with either cisplatin, doxorubicin or RHl.

[0042] Table 1.

[0043] Table 1 shows IC50 values obtained from this experiment for each cell line.

(C) indicates cisplatin, (D) indicates doxorubicin, and (R) indicates RH 1. It is noted that IC50 values for RHl for the tumor types indicated varied between 1.5 nM and 7.5 nM, which is an order of magnitude less than values for doxorubicin and two orders of magnitude less than cisplatin. It is also noted that low (nM scale) IC50 values were obtained for all cell lines, whether or not they appeared to express DTD, leading to the conclusion that there is no correlation between clonogenic survival and DTD expression in RHl treated cells. See Figure 2, from which the data in Table 1 is obtained.

Example 3:

[0044] Figure 3 shows survival percentage of all cell lines obtained using the SRB assay 5 days after 1 hour treatment with cisplatin, doxorubicin, or RHl. Figure 3 shows the data obtained, and Table 2 summarizes the data obtained.

[0045] Table 2.

[0047] Table 2 shows IC50 values obtained from thi

(C) indicates cisplatin, (D) indicates doxorubicin, and (R) indicates RH 1. It is noted that IC50 values for RHl for the cell lines indicated varied between 1 nM and 200 nM. In this particular assay, sensitivity to RHl(SRB assay) correlated with DTD expression (DTD

expression shown in Example 1), except for RDES cells. However, it is noted that all cell lines exhibited sensitivity to RHl that was greater than either cisplatin or doxorubicin, and sensitivity to RHl was on a nM-scale.

Example 4:

[0048] Figure 4(A) shows Western blots for full-length (116 kD) and cleaved (85 kDa) PARP (poly ADP ribose polymerase, involved in DNA repair and apoptosis) after exposure to RHl at various concentrations for one hour. For each cell line tested, the IC50 values obtained from the experiment in Example 3 (SRB assay) were used as an intermediate value. Figure 4(B) shows bar graphs plotting the percentage of apoptotic cells 24 hours after treatment with RHl (1 hour), using Hoescht stained drug-treated cells. Each value represents the mean and standard error derived from two independent experiments in which at least 100 cells were counted. This data shows increased PARP cleavage and apoptosis in response to RHl in cell lines expressing DTD (791T and LA1-5-S).

Example 5:

[0049] From Example 3, it was noted that Ewing's sarcoma cell lines A673 (noted as a positive expressor of DTD) and RDES (noted as a non-expressor of DTD) had very little differential sensitivity to RHl in the SRB assay (IC50 of 38 and 28 nM, respectively). In Example 5, PARP cleavage was measured as discussed in Example 4. Table 5(A) shows that A673 and RDES show similar PARP cleavage in response to RHl, correlating with lack of difference in IC50 in the SRB assay. Figure 5(B) shows bar graphs plotting the percentage of apoptotic cells 24 hours after treatment with RHl (1 hour), using Hoescht stained drug treated cells, showing little difference between A673 and RDES cells' apoptotic responses to RHl, also correlating with lack of differential sensitivity for these two cell lines in SRB assay.

Example 6:

[0050] Figure 6 shows apoptosis induced by RHl in A673 Ewing's sarcoma xenografts in nude mice, after 0.4 mg/kg single IP injection of RHl. Bars show immunohistochemistry for cleaved caspase 3 at specific times after treatment, showing the largest effect at 24 hours.

Example 7:

[0051] Figure 7 shows a comparison between control, cisplatin, and RHl-treated

A673 Ewing's sarcoma xenografts in nude mice, mean volume at day 10 after drug treatment (0.4 mg/kg daily for 5 days). Each data point represents an individual mouse. RHl demonstrates inhibition of growth of this tumor in this model, particularly in comparison with cisplatin.

Example 8:

[0052] Figure 8 shows a comparison of the effect of RHl on the growth of A673

Ewing's sarcoma (8A) and 79 IT osteosarcoma (B) xenografts in nude mice. RHl was given 0.4 mg/kg daily by IP injection for 5 days after tumors reached 200 ml in volume. Control mice received DMSO injections. Plots show estimated mean slope. Statistical analysis of the differences in slope using the mixed linear effects model gives p=0.19 for A673 cells and p=0.10 for 79 IT cells. Pooled analysis between the two experiments gives p=0.05 for the differences in mean slope between RHl treated mice and control.

Example 9:

[0053] Figure 9 shows a comparison between control, cisplatin and RHl-treated

A673 Ewing's sarcoma xenografts in nude mice, time to tumor volume greater than 1000 ml after drug treatment (0.4 mg/kg IP daily for five days).

Example 10:

[0054] In this example, cell lines discussed in Example 1 were tested for whether combination treatment with RHl/cisplatin or RHl/doxorubicin was synergistic. Figure 10(A) and 10(B) shows combination index values for the effective dose at 50% (ED50), 75% (ED75) and 90%(ED90) for a combination treatment of RHl with cisplatin (10A) or doxorubicin (10B) for one hour, using CALCUSYN (available from Biosoft, Inc.) Data indicates that synergy exists with cisplatin for at least LA1-55N (neuroblastoma) and RDES (Ewing's sarcoma), suggesting that treatment of at least these cancers with a combination of RH 1 and cisplatin may be more effective than either alone. Data indicates that synergy exists with doxorubicin for at least LA1-55N (neuroblastoma) and U2OS (osteosarcoma), suggesting that treatment of at least these cancers with a combination of RHl and doxorubicin may be more effective than either alone.

Example 11 :

[0055] Chemical Synthesis of RHl (2,5-diaziridinyl-3-hydroxymethyl-6- methyl- 1,4 benzoquinone) may be synthesized as follows.

[0056] To a stirred solution of 2-hydroxymethyl-5 -methyl- 1,4 benzoquinone (1O g,

65.8 mmol) in ethanol (250 ml), under N2 at 0 ⁰ C, was added aziridine (6. 8 ml, 5.66 g, 131.6 mmol). After 20 minutes the solution was allowed to rise to room temperature and stirred for a further 5 hours. The solvent was then reduced in vacuo to approximately 100 mis and then cooled on ice. The resulting precipitate was filtered and washed with ice cold ethanol (50 ml). A further crop could be obtained by reducing the solvent to about 50 ml, cooling and filtering again. The combined yield was 2.813 g of dark red crystals. (18.3%, m.p. 178-9° C); ¹ H NMR (200 MHz, CDCl ₃ ): 64. 56 (2H, d, J=6 Hz, CH ₂ ), 2.64 (IH, t, J=6 Hz, OH), 2.38 (4H, s, Az), 2.28 (4H, s, Az), 2.0 (3H, s, CH ₃ ); MS El m/z: 234 (M+), 219, 191, 177, 163, 149; V _max (KBr disc): 3483, 2995, 1637, 1585, 1383, 1300, 1159; HREIMS. Found 234.1005 C ₂ Hi ₄ N ₂ O ₃ requires 234.1004.

[0057] RHl is easily synthesized with very high purity (99%). RHl is readily soluble in aqueous solution (solubility in phosphate buffered saline is (0.5mg/ml at 25°C). The RHl solutions are very stable with a half life of RHl in phosphate buffer (0.1 to 1 M, pH=7) of more than 2 days at 25°C The free hydroxyl group of RHl accounts for its water solubility that leads to a shorter half-life in pharmacokinetics.

[0058] The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limiting of the invention to the form disclosed. The scope of the present invention is limited only by the scope of the following claims. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment described and shown in the figures was chosen and described in order to best explain the principles of the invention, the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.