The present invention relates to non-radioactive enzymatic methods for detecting Sphingosine-1-Phosphate (S1P) in biological fluids. The present invention further relates to a method of detecting the presence of cancer in a patient by the use of these and other methods of detecting (S1P) in biological samples from a patient.

The present invention relates to non-radioactive enzymatic methods for detecting Sphingosine-1-Phosphate (S1P) in biological fluids and the correlation at a measurement of S1P to disease. The present invention further relates to a method of detecting the presence of cancer in a patient by the use of these and other methods of detecting S1P in biological samples from a patient.
Sphingolipids are a diverse group of molecules that are found in the membranes of all eukaryotic cells (Merrill et al., Toxicol. Appl. Pharmacol. 142:208-225 (1997)). There are three classes of sphingolipids: sphingomyelins, cerebrosides and gangliosides. Sphingolipids include sphingomyelin, sphinganine, sphingosine, glycosphingolipids and ceramide. Sphingosine-1-phosphate (S1P) is a lysosphingolipid that is generated by the metabolism of sphingomyelin. (Spiegel and Merrill, FASEB J. 10:1388-1397 (1996)). Sphingomyelin, the most abundant type of sphingolipid is first converted to ceramide (sphingosine+palmitate residues) by the removal of the head group. Ceramide is then cleaved to form sphingosine by the action of ceramidases. S1P is then generated from sphingosine by the action of sphingosine kinase. S1P may then be further metabolized into ethanolamine-phosphate and long-chain aldehyde (e.g. hexadecanal) by S1P lyase (Zhou and Saba, Biochemical and Biophysical Research Communications, 242:502-507 (1998)). The web of enzymatic events governing the metabolism of Sphingolipids is diagrammed below:
In addition to their role as components of biological membranes, some of the metabolites of sphingolipids, such as ceramide, sphingosine and S1P, are potent chemical messengers (Spiegel and Merrill, supra, Meyer et al., FEBS Lett. 410:34-38 (1997); and Gomez-Munoz et al., J. Biol. Chem. 270:26318-26325 (1995)). S1P has been shown to act as an intracellular second messenger that is generated in cells that have been activated by various mitogens (Spiegel et al., Breast Cancer Res. Treat. 31:337-348 (1994); Sadahira et al., Proc. Natl. Acad. Sci. USA 89:9686-9690 (1992); and Spiegel et al., Ann. N.Y. Acad. Sci. 845:11-18 (1998)), and to act as an extracellular ligand for a group of cell surface receptors (Hecht et al., J. Cell. Biol. 135:1071-1083 (1996); An et al., Biochem. Biophys. Res. Commun. 231:619-622 (1997); Goetzl and An, FASEB J. 12:1589-1598 (1998); van Koppen et al., J. Biol. Chem. 274:18997-19002 (1999); Yatomi et al., J. Biol. Chem. 272:5291-5297 (1997); and Ancellin and Hla, J. Biol. Chem. 274:18997-19002 (1999)). S1P is the principal ligand for three G-protein coupled receptors known as endothelial cell differentiation gene-1, -3 and -5 (Edg-1, Edg-3 and Edg-5). Edg-1, -3 and -5 are expressed in a variety of human tissues. Edg-1 is ubiquitously expressed (Goetzl and An, supra). Edg-3 is abundant in cardiovascular tissue and leukocytes, and is also expressed widely (Id.). Edg-5 is found most abundantly in cardiovascular tissue, the central nervous system, gonadal tissue and the placenta (Id.). The presence of multiple, high affinity receptors for S1P and their wide distribution, may provide redundancy in the control of the biological processes mediated by S1P. Another possibility is that specific S1P-directed biological processes are achieved by differential coupling between the various receptors and G-proteins (Ancellin and Hla, supra). However, the full role of S1P in normal and abnormal cell function has not yet been elucidated.
Previously, S1P levels have been measured quantitatively in biological samples by various methods utilizing the addition of radioactive isotopic moieties to the molecule. One such method is the determination of the amount of radioactive acetic anhydride that is incorporated in S1P by acylation (Yatomi et al., Anal. Biochem. 230:315-320 (1995)). In this assay, the S1P is first extracted from cells into an upper aqueous phase under alkaline conditions, and then re-extracted into the lower chloroform phase under acidic conditions. The phosphorylated sphingoid base is then quantitatively converted to N-[3H]-acetylated S1P by N-acylation with [3H] acetic anhydride, forming C2-Cer-1-P (tritiated C2-ceramide 1 -phosphate). The C2-Cer- 1-P is resolved with thin-layer chromatography, detected with autoradiography, and quantified with a scintillation counter. The assay permits quantification of S1P over a range of at least 100 pmol to 10 nmol. Yatomi used this assay to measure the distribution of S1P in various organs in rats. The largest concentration of S1P was in the testis and intestine (xcx9c100 nmol S1P/gram of tissue), followed by the spleen and brain (xcx9c50 nmol S1P/gram of tissue) (Yatomi et al., FEBS Lett. 404:173-174 (1997)). The kidney, heart and lung contained between 10 and 20 nmol S1P/gram of tissue, with the liver and muscle containing the lowest concentration of S1P ( less than 5 nmol S1P/gram of tissue).
Using their assay, Yatomi and coworkers measured the level of S1P in some human bodily fluids (Yatomi et al., J. Biochem. (Tokyo) 121:969-973 (1997)). They determined the concentration of S1P in normal human plasma and serum, but were unable to detect S1P using their assay in urine, ascites, pleural effusion or cerebrospinal fluid. They determined that tritiated sphingosine was rapidly taken into platelets in platelet-rich serum, while S1P was stable, demonstrating that the enzymes required for S1P degradation are not present in serum (Yatomi et al., J. Biochem. (Tokyo), supra). The authors suggested that the release of S1P from activated platelets might be involved in processes such as thrombosis, hemostasis, atherosclerosis and wound healing.
S1P has also been detected using an enzymatic method employing an alkaline lipid extraction to separate S1P from other phospholipids and sphingolipids (Edsall and Spiegel, Anal. Biochem. 272:80-86 (1999)). The extracted S1P was converted to sphingosine by alkaline phosphatase treatment. The sphingosine was then quantitatively phosphorylated using recombinant sphingosine kinase and [xcex332P]ATP. The authors reported that levels of S1P varied in rat tissues between 0.5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while the brain contained the highest levels.
These prior known methods for measuring S1P focus on the incorporation of a radioactive isotopic moiety into the molecule, and require considerable processing time in the laboratory. These limitations make the current assays unsuitable for many clinical or laboratory settings where complex bench processes are not practical. Development of a rapid and sensitive non-radioactive assay for S1P would facilitate use of this compound as a marker for various cellular activities such as cell growth, cell migration, apoptosis, platelet activation, changes in cellular morphology and for detecting conditions associated with altered levels of S1P.
Cancers such as ovarian cancer, lung cancer, colon cancer, and breast cancer are among the most frequent causes of cancer death in the United States and Europe. Despite decades of cancer research, mortality rates among persons who contract cancer remain high. However, when a cancer is detected at an early stage, survivability increases dramatically. For example, when ovarian cancer is diagnosed at an early stage, the cure rate approaches 90%. In contrast, the 5 year outlook for women with advanced disease remains poor with no more than a 15% survival rate. Thus, early diagnosis is one of the most effective means of improving the prognosis for cancer.
Frequently, however, detection of cancer depends upon the detection and inspection of a tumor mass which has reached sufficient size to be detected by physical examination. The detection of molecular markers of carcinogenesis and tumor growth can solve many of the problems which the physical examination of tumors have encountered. Samples taken from the patient for screening by molecular techniques are typically blood or urine, and thus require minimally invasive techniques. Thus, they can be used on a regular basis to screen for cancers. In addition, because molecular markers often appear before the tumor is of a detectable size, it is possible to detect cancers at very early stages in the progression of the disease.
The present invention provides a molecular marker for screening and diagnosis of disease while overcoming the deficiencies in the current methods of measuring S1P by providing simple enzymatic methods for determining concentrations of S1P in samples of biological fluids such as serum or plasma. The methods utilize the enzymatic cleavage of S1P by to produce ethanolamine-phosphate and long-chain aldehyde (e.g. hexadecanal), and a separate second enzymatic step which produces a detectable signal that can be used to calculate the concentration of S1P in the sample.
In the various embodiments of this aspect of the invention, the concentration of S1P is detected in a sample of bodily fluid taken from a patient. The S1P in the sample is preferably first enriched through extraction of sphingolipids. S1P in the sample is digested in the first enzymatic step with S1P lyase, to generate long-chain aldehyde (e.g. hexadecanal) and ethanolamine-phosphate. In the second step of the enzymatic detection aspect, either S1P or one of the products of the enzymatic cleavage is utilized as a substrate by an enzyme that produces a product which can be detected by chemiluminescent, fluorescent, spectraphotometric or calorimetric methods the second step. In more preferred embodiments of the invention, an oxidation-reduction reaction is utilized in the second step to produce a detectable signal via enzymes that amplify the signal which is detected by chemiluminescent, fluorescent, spectraphotometric or colorimetric methods the second step. In preferred embodiments of the invention, a portion of the sample extract which is not subjected to the first enzymatic step is subjected to the second enzymatic step as a control to accurately measure the increase or decrease in the signal caused by the digestion of S1P by S1P lyase in the first enzymatic step.
Another embodiment of the S1P detection methods of the present invention is a diagnostic kit containing enzymes and other reagents for conducting the enzymatic assays of the invention to measure the concentration of S1P in samples of bodily fluids taken from patients.
In another aspect of the present invention, altered levels of S1P in a sample of bodily fluid taken from a human patient, as compared to normal levels of S1P, are used to detect cancer in the patient. The enzymatic methods of the invention, as well as other methods of measuring S1P, may be utilized in this aspect of the invention. The determination of abnormal S1P concentration may be made by comparison with a numerical or experimental standard or by determining a change in the S1P level of a patient over time. The method of the present invention can be used to detect a broad range of cancers at an early stage. Gynecological cancers, including breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, peritoneal cancer, and fallopian tube cancer, are particularly suitable for detection using the methods of the present invention.
In alternative embodiments of the cancer detection method of the present invention, the concentration of additional markers for cancer in the sample of bodily fluid are also determined, either sequentially or simultaneously. By testing for additional cancer markers, the clinician can increase the accuracy and specificity of a diagnosis based upon the an abnormal level of S1P in the patient""s sample. Also, by conducting repeat measurements over time, the clinician can monitor the progression of disease or response to therapy by correlating the change in S1P levels over time to previous values or controls.
In the enzymatic detection methods of the invention a bodily fluid sample such as whole blood is collected from a patient. The patient may be any eukaryotic organism, preferably a vertebrate, including, but not limited to, a mammal, a bird, a fish, an amphibian, or a reptile. Preferably, the patient is a mammal, most preferably a human. Bodily fluids include, but are not limited to, plasma, serum, urine, saliva, ascites, cerebral spinal fluid or pleural fluid. As mentioned above, the enzymes necessary to specifically degrade S1P are not found in whole blood. Such stability may facilitate the measurement of S1P in blood products. Thus, whole blood derivatives such as serum or plasma are preferred for the use in the present invention to monitor S1P levels in a patient. However, the methods of the present invention may also be utilized to measure S1P levels in other biological samples if care is taken to minimize degradation of the analyte. General enzymatic inhibitors known in the art, such as E-64 (trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane, Sigma); leupeptin (Sigma); pepstatin A (Sigma); TPCK (N-tosyl-L-phenylalanine chloromethyl ketone, Sigma); PMSF (Phenylmethanesulfonyl fluoride, Sigma); benzamidine (Sigma) and 1,10-phenanthroline (Sigma), EDTA (Ethylenediaminetetracetic acid, Sigma) and EGTA (Ethylene glycol-bis-(beta-aminoethyl ether), Sigma) may be used in the collection of such samples. EDTA may also activate the S1P lyase enzyme in the enzymatic detection method.
In order to increase the amount of S1P in the sample, sphingolipids are preferably extracted from the sample. For example, organic extraction using chloroform:methanol and centrifugation can be used to enrich for S1P, as described in Example 1. This organic extraction may be altered by one skilled in the art to optimize for S1P extraction (e.g. acidic or basic conditions). In the methods of the invention, an alternative to the liquid organic extraction for enrichment includes the use of solid phase extraction using, e.g. a Bond-Elut7 column (Varian, Harbor City, Calif.) consisting of silica or fluorosil can be used to enrich for S1P and to remove proteins and other lipids.
In the preferred embodiments of the invention, S1P is hydrolyzed by S1P Lyase, (Zhou and Saba, supra) into a mixture of ethanolamine-phosphate and long-chain aldehyde (e.g. hexadecanal) in the first enzymatic step. This substrate-specific enzymatic reaction allows for the detection of S1P in the second enzymatic step by either the disappearance of S1P as a substrate for a non-substrate-specific enzymatic reaction, or the appearance of ethanolamine-phosphate or long-chain aldehyde (e.g. hexadecanal) as a substrate for a specific or non-specific enzyme reaction (such as the utilization of long-chain aldehyde, e.g. hexadecanal, by bacterial luciferase in a chemiluminescent reaction.) Although it is preferred that S1P, ethanolamine-phosphate, or long-chain aldehyde (e.g. hexadecanal) be utilized as a substrate by an enzyme which produced a product which may directly be detected by chemiluminescent, fluorescent, or spectraphotometric means, embodiments where the first product produced is an intermediate product/substrate for later enzymatic reactions which produce a detectable product are also envisioned within the present invention. The second enzymatic step preferably comprises additional enzymes to recycle substrates in order to magnify the signal obtained from the utilization of S1P, ethanolamine, or long-chain aldehyde (e.g. hexadecanal) as a substrate.
In preferred embodiments of the invention, the second enzymatic step comprises a luminescent reaction to detect the amount of long-chain aldehyde (e.g. hexadecanal) produced by generating a light signal, which is then detected, for example, using a luminometer. This luminescent reaction utilizes two enzymes, NAD(P)H:FMN oxidoreductase and luciferase, to produce light signal (Ugarova et al., Anal. Biochem. 173:221-227 (1988); Dunn, et al., Biochemistry 12(24): 4911-4918 (1973); Lavi, et al., Biochemical and Biophsical Resrch. Communications, 111(1): 266-273 (1983)). These enzymes catalyze the following reactions:
NAD(P)H+FMN+H+xe2x86x92FMNH2+NAD(P)+xe2x80x83xe2x80x83(1)
FMNH2+O2+RCHOxe2x86x92FMN+RCOOH+H2O+hvxe2x80x83xe2x80x83(2)
(1) NAD(P)H:FMN oxidoreductase reaction
(2) Luciferase reaction
All of the components of the above enzymatic method are added except for long-chain aldehyde (RCHO) which is supplied by the S1P lyase reaction with S1P in the first step. The level of S1P detected is quantified by detecting the luminescence (counts/second) measured by a luminometer (Wallac Victor2 Multilabel Counter, EGandG Wallac, Inc., Gaithersburg, Md.). The difference between the amount of light produced by the luciferase reaction without the addition of S1P lyase in the first enzymatic step of the assay and the amount produced with the addition of S1P lyase is directly proportional to the amount of S1P present. Numerical values are obtained from a standard curve consisting of known amounts of long-chain aldehyde (e.g. hexadecanal from 0 to 100 xcexcM), or by subjecting known amounts of S1P to the first and second enzymatic steps. Assays are preferably performed in duplicate with both positive and negative controls. Background signals in plasma without S1P Lyase are subtracted from all samples. Standard curve values are plotted and fitted to a point-to-point, linear or second-order polynomial curve fit. The levels of S1P in each sample are determined by comparing each signal measured to the standard curve.
In another embodiment, the second enzymatic step utilizes an aldehyde dehydrogenase reaction in the presence of NAD+ to produce a long-chain acid and NADH:
xe2x80x83RCHO+NAD(P)++H2Oxe2x86x92RCOOH+NAD(P)Hxe2x80x83xe2x80x83(1)
(1) aldehyde dehydrogenase reaction
The level of S1P detected is quantified by detecting the production of NAD(P)H using UV spectrometry at OD340, colorimetry, fluorescence, or luminescence by standard procedures known in the art. For this reaction, several types of aldehyde dehydrogenase enzymes may be sufficient including NAD-dependent (EC 22.214.171.124) and NAD(P)-dependent (EC 18.104.22.168) enzymes. These enzymes generally interact with short-chain aldehydes. S1P molecules generally contain long-chain sphingosine, and therefore, would result in long-chain aldehydes after S1P lyase treatment. If necessary, a long-chain aldehyde dehydrogenase (EC 184.108.40.206) may be used in this reaction. Use of long-chain aldehyde dehydrogenase may reduce background signal from non-specific short-chain aldehydes and be useful for enzymatic determinations of S1P in unextracted plasma or serum.
In another embodiment, the second enzymatic step utilizes a cycling reaction to amplify the long-chain aldehyde (e.g. hexadecanal) signal. In this cycling reaction, two types of aldehyde dehydrogenase enzymes are used. Aldehyde dehydrogenase enzymes that specifically require NADH or NADPH as cofactors are combined to cycle between long-chain aldehydes and long-chain fatty acids. For example, NAD-dependent aldehyde dehydrogenase and NADxe2x88x92 are utilized to catalyze long-chain aldehyde to long-chain fatty acid. NADP-dependent aldehyde dehydrogenase and NADPH are utilized to catalyze long-chain fatty acid to long-chain aldehyde.
xe2x80x83RCHO+NAD++H2Oxe2x86x92RCOOH+NADHxe2x80x83xe2x80x83(1)
(cycling)
RCHO+NADP++H2O←RCOOH+NADPHxe2x80x83xe2x80x83(2)
(1) NAD-dependent aldehyde dehydrogenase reaction
(2) NADP-dependent aldehyde dehydrogenase reaction
Sufficient amounts of NAD- and NADPH are added to this cycling reaction to drive the (1) reaction to the right and the (2) reaction to the left as shown. These reactions are allowed to cycle until sufficient signal is generated. The level of S1P detected is quantified by detecting the production of NADH or the disappearance of NADPH using detection systems specific for NADH or NADPH. One such detection system utilizes a similar luminescent reaction described above. In this reaction, FMN:NAD(P)H oxidoreductases specific for either NADH or NADPH (Jablonski and DeLuca, Biochemistry, 17(4): 672-678 (1978)) are used with luciferase to generate light. This reaction is similar to the bacterial luciferase luminescent reaction described above, however the NADH accumulated or NAD(P)H oxidized from the above cycling reaction is measured instead of the hexadecanal produced by S1P lyase cleavage. For this detection reaction, no additional NADH is added and an excess of long-chain aldehyde reagent is added after the above enzymatic cycling reaction is complete. As a result, the luminescent reaction determines the NADH concentration accumulated in the above enzymatic cycling reaction, which will be proportional to the original concentration of hexadecanal produced in the S1P lyase cleavage reaction.
The above two examples of second enzymatic step detection reactions merely illustrate the principle of the invention. One of ordinary skill in the art would similarly be able to utilize any number of known reactions for the measurement of aldehyde in the sample in order to detect the appearance of long-chain aldehyde (e.g. hexadecanal). Alternatively, other reactions could be utilized to detect the disappearance of a reactive group on S1P or the appearance of a reactive group on ethanolamine-1-phoshate. Thus, by utilizing the substrate-specific S1P lyase reaction, the quantification of S1P in the sample can be achieved by monitoring the subtractive or additive difference in the signal produced by the non-substrate specific second enzymatic step. Because an the detection methods utilize enzymatic reactions to produce a detectable signal, they may be carried out in a reasonable amount of time for use in clinical screening and large-sample-number research settings.
An important advantage of the enzymatic assay of the invention is that the assay may be performed in a microtiter plate format to permit small volumes of samples and reagents to be employed. In addition, the results of the assay may be monitored with automated equipment, e.g. using an ELISA reader. These formats facilitate automating the performance of the assay. Reduced processing times and automated handling using such formats for the assays may reduce variability between results.
The methods of the invention provide a rapid and accurate assay with increased sensitivity for detecting small amounts of S1P present in samples of bodily fluids from patients. The enzymatic assay can be used to detect conditions associated with altered levels of S1P in a sample from a patient as compared to normal samples. The methods of the invention and test kits thus provide a practical means for clinical detection of conditions associated with altered levels of S1P and the monitoring of S1P levels in patients, such as neurodegenerative diseases or the novel detection of cancer described below. In addition, the enzymatic assay of the invention is an important research tool for the exploration of the role of S1P in normal biology and various pathologies.
The enzymatic methods described herein for measuring concentrations of S1P in samples of bodily fluids from a patient may also be performed, for example, by using pre-packaged diagnostic kits. Such kits optionally include components such as enzyme S1P lyase, enzyme reagents for carrying out the second step of the methods of the invention, (e.g. bacterial luciferase, NAD(P)H:FMN oxidoreductase, NADH or NAD(P)H specific aldehyde dehydrogenases, etc.). Also optionally included are ancillary agents such as buffering agents, substrate reagents such as NADH or NAD(P)H, and agents such as EDTA to inhibit subsequent production or hydrolysis of S1P during transport or storage of the samples. Kits embodying the enzymatic method aspect of the invention may also include control standards such as long-chain aldehyde (e.g. hexadecanal), S1P, or ethanolamine-phosphate in defined concentrations. The kits may also include an apparatus or container for conducting the methods of the invention and/or transferring samples to a diagnostic laboratory for processing, as well as suitable instructions for carrying out the methods of the invention. If the kit is to be used in the cancer detection method of the invention, the kit may also optionally include components for the detection of other cancer markers as described below.
Detection of Cancer in Patients by Detection of Abnormal Levels of S1P
The methods of the invention can be used to provide a rapid and economical screen of large numbers of patients to promote early diagnosis of cancer associated with altered levels of S1P. Early detection provided by such methods can significantly improve quality of life and better patient survival rates by permitting the early and aggressive management of the disease at its nascent stages.
In preferred methods of the cancer detection aspect of the invention, the S1P level of the patient is determined or monitored utilizing the enzymatic detection aspect of the invention described above. However, it is also envisioned that alternative methods of measuring S1P in a bodily fluid sample obtained from the patient may be employed in the cancer detection aspect of the invention. For instance, S1P in the biological sample may be measures by fractionating the sample using HPLC or gas chromatographic techniques, or purification/extraction procedures, and then quantified on a mass-spectrometer or by spectraphotometry. Alternatively, antibodies reactive with S1P may be generated using standard methods, for example by immunizing a suitable animal with S1P and an adjuvant to produce antibodies and the fusion and immortalization of spleen cells (prepared as described by Kohler and Milstein (Nature 256:495-497)), or by recombinant methods, as are generally known. These monoclonal or polyclonal antibodies may then be used in an assay such as a radio-immunoassay (RIA), enzyme linked immunoassay (ELISA), or other immunoassay formats to detect the presence of S1P in a sample of bodily fluid.
The specific correlation of the level of S1P in the sample with the cancer state is usually specific for the type of sample and type of cancer. For instance, in a plasma sample the level of S1P may be greater than in a serum sample for the same patient with the same cancer condition. In addition, one type of cancer may show a decrease in S1P concentration as compared to normal levels, while another cancer type may show an increase in S1P concentration. Likewise, the use of different extraction procedures to enrich the amount of S1P in the sample will alter the absolute value of the diagnostic threshold. The person of ordinary skill in the art would be capable of determining the proper level of S1P in the sample which is indicative of a particular disease state, given the guidance supplied by this specification and the examples below, utilizing routine experimentation. For instance, one of ordinary skill in the art would know to first establish an positive indicator threshold level of S1P for a particular sample technique (serum, plasma, or urine) by first comparing samples taken from normal patients with those diagnosed as having the particular cancer (breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, peritoneal cancer, and fallopian tube cancer.) Alternatively, normal or control values may be obtained, when known, from published literature. By making a comparison between normal or control values and those indicative of disease, utilizing samples available from various specimen banks and the assay techniques detailed below, one may establish the proper indicative threshold to diagnose a patient as having a particular type of cancer. In general, a finding that a patient has a level of S1P in his or her biological fluids, which deviates significantly from the normal range is a strong indication that the patient has cancer.
In addition to its use as a detection method, the response of a cancer condition to treatment may be monitored by determining concentration of S1P in samples taken from a patient over time. The concentration of S1P is measured and compared to the concentration taken at the earlier time from that patient. If there is a significant change in the concentration of S1P over time, it may indicate an increase in the severity of the condition in the patient. Conversely, if there is a normalization of the concentration of S1P, it may indicate an improvement in the condition of the patient.
The methods disclosed herein are simple, marginally invasive, and require only a specimen such as blood or urine from the patient. Thus, such methods are also useful for screening patients who have not been previously diagnosed as carrying cancer, particularly patients who are at risk for cancers, especially ovarian cancer or breast cancer. Such patients include women at elevated risk by virtue of a family history of the disease, premenopausal women with anovulatory cycles, and postmenopausal women.
In addition to the detection of levels of S1P, to improve the sensitivity and/or the specificity of detection of disease, the methods of the invention may include detection of levels of other compounds, such as other sphingolipids including sphingomyelin, sphinganine, sphingosine, glycosphingolipids and ceramide, which are detectable, e.g., by mass spectroscopy (Veldhoven, et al., FEBS Letters, 350: 91-95 (1994)). Alternatively, or in addition, the level of lysophospholipids including the concentration of total lysophospholipid in a sample, and/or the concentration of specific types of lysophospholipids including lysophosphatidic acid (LPA), lysophosphatidyl choline (LPC), lysophosphatidyl serine (LPS), lysophosphatidyl ethanolamine (LPE), lysophosphatidyl inositol (LPI) and lysophosphatidyl glycerol (LPG) and/or the levels of glyerol-3-phosphate and its phosphatidyl derivatives may also be measured according the methods described in PCT/US99/24969 or PCT/US98/05738, which are specifically incorporated herein.
Alternatively, or in addition, proteinaceous cancer markers may be detected in conjunction with S1P in a patient. Such markers include, but not are limited to, CA125 (detectable by the method of Taylor, supra), Tac (detectable by antibody as per U.S. Pat. No. 4,545,985), soluble IL2 receptor alpha (detectable by antibody as per U.S. Pat. No. 5,351,772), mCSF(detectable by antibody as per Saitoh, et al., J. Am. Coll. Cardiology, 35(3):655-65 (March 2000)), OVX1 (detectable by antibody as per Martell, et al., Intl. J. Biol. Markers, 13(3):145-9 (1998), CEA (detectable by antibody as per Wichmann, et al., Langenbecks Arch. Surgery, 385(4):271-5 (July 2000)), PSA (detectable by antibody as per Koch, et al., J. Urology, 164:749-53 (September 2000)), CA15-3 (detectable by antibody as per Dimas, et al., Euro. J. Gynaecology and Oncology, 21(3):278-81 (2000)), CA19.9 (detectable by antibody as per Kamoshida, et al., Modern Pathology, 13(7):736-41 (July 2000), and PLA2 (detectable by immunoassay as per U.S. Pat. No. 5,747,264).
The measurements of additional sphingolipids and/or lysophospholipids and/or proteinaceous cancer markers may be used in combination with the level of S1P detected to refine the detection of disease. As many of these compounds may be measured in a bodily fluid sample from a patient, especially plasma or serum, measurement of these markers may be easily carried out with a portion of the bodily fluid sample taken from the patient concurrently with the determination of S1P. These additional measurements may reduce the false positives and negatives associated with detection of disease in a patient using the methods of the invention. Measurements of different markers can be taken either simultaneously or sequentially from a single sample from a patient. Particularly useful measurements for increasing sensitivity are measurements of concentrations of S1P and other markers taken over time or in units of rate of change of the marker over time, to decrease false positive results.
The information on levels of S1P in samples from a patient made available by the methods of the invention may suggest additional procedures be instituted to improve the early detection of diseases such as cancer, such as ultrasound, biopsy, laparascopy, surgery, mammography, biopsy or MRI.

Enzyme method for detecting sphingosine-1-phosphate (S1P)


Structured antiperspirant emulsion formulations for topical application to human skin in a cosmetic method for controlling sweat and body odor generation can suffer from problems of impaired sensory properties and impaired efficacy (sweat reduction), which are ameliorated or overcome in structured antiperspirant emulsions in which (%s by weight of the emulsion)

The present invention relates to antiperspirant formulations for topical application and in particular to formulations in the form of emulsions.
Humans perspire aqueous sweat through eccrine glands in the course of exercise or as a consequence of emotional stress. In certain areas of the body such as underarm, there is an especially high surface density of eccrine glands which can result in sweat becoming visible, either on the skin itself or by absorption by clothing which comes into contact with the skin in the vicinity of the armpits. In many societies, people wish to avoid the sweat being visible, at least on some areas of the body such as underarm, and accordingly apply topically antiperspirant formulations to those selected areas.
Many antiperspirant formulations which have been developed or/and commercialised comprise an astringent salt, such as an astringent aluminium and/or zirconium salt in a suitable carrier system. Applicators for the formulations can be classified as either contact or non-contact applicators. Non-contact applicators are especially hygienic because there is no contact between the dispenser and the human body in the vicinity of where the formulation is to be applied, but many consumers desire to use contact applicators, especially in North America.
Formulations for contact applicators can themselves be classified into three broad types, based on their physical characteristics. One type comprises lotions, which flow under gravity and are commonly dispensed using a roll-on applicator. A second type comprises a cream or soft solid which commonly have sufficient inherent structure that they either flow very slowly or retain their shape when they are not subjected to pressure, but which flow under pressure. These are commonly dispensed through an applicator comprising a container for the formulation, a perforated cap for the container through which the formulation can flow and a means to pressurise the formulation and urge it towards the perforated cap. A third type comprises a firm stick, which is structured sufficiently to maintain its integrity even when subjected to gentle pressure.
The structured formulations can themselves be further classified into three classes. One class comprises solution formulations. As the name suggests, the active constituent is dissolved in the carrier fluid. In a second class, which comprises suspension formulations, the active constituent is not soluble in the carrier fluid and is present in the form of suspended particles. In the third class, the formulation comprises an emulsion in which droplets of one liquid phase, the internal phase, are dispersed within a continuous second liquid phase, the external phase. Commonly, in respect of antiperspirant formulations, one of the phases is hydrophobic and the other is hydrophilic and the active material is dissolved in the hydrophilic phase. The instant invention relates particularly to the third class, namely structured formulations in the form of such emulsions.
Consumers have regard to many different aspects of antiperspirant products when making their choice from which are products available. These include a choice between contact and non-contact applicators, the physical attributes of an antiperspirant formulation, such as lotion, cream or firm stick and whether it is efficacious. However, they have regard to the aesthetics of the product, such as its appearance and the skin sensations on application of the product, especially when contemplating a repeat purchase. In other words, they take into the account the sensory and visual characteristics of the product. The various different sensory characteristics include whether the formulations feels cool or not on application, whether it feels dry or wet on application, whether it feels sticky, the smoothness with which a contact formulation glides across the skin and whether it feels greasy.
It is a continuing objective of producers of antiperspirant formulations to meet the evolving needs or preferences of their consumers. In that context, the inventors have carried out further investigations into structured antiperspirant emulsions. It will be recognised that emulsions represent comparatively complex systems for delivering an antiperspirant to the skin with inter-relationships between various of the formulation constituents. In common with non-emulsion formulations, there are constraints on the proportions of the various components which can be employed. For example, increasing the proportion any particular constituent reduces the formulation headspace for other constituents, but can also impact upon the efficacy and aesthetics of the product.
However, for emulsions, there is additionally the complication of there being two liquid phases, in both of which their constituents can be varied as well as the volume or weight ratio between the two of them being varied. It will also be recognised that the antiperspirant in an emulsion is commonly a constituent of a disperse hydrophylic phase. The inventors have identified that in order for the antiperspirant active to be effective, the disperse phase should be brought into close contact with the skin in the vicinity of its sweat-producing eccrine glands. The inventors have further found that varying the constituents of one phase may not only change a target functional attribute, but at the same time may also change either the aesthetics or appearance of the product as well, possibly in a manner which is less attractive to consumers. For example, changing the proportion of antiperspirant active solution in the formulation not only can have the effect of changing the efficacy of the product, but can simultaneously change the feel of the product and changing the relative or absolute amounts or the nature of constituents of either phase can alter the appearance of the product. Consequently, the inventors have not only found that there are several relationships between the components of structured emulsions which should be taken into account when seeking to produce an emulsion product, but also that to at least some extent, they can conflict with each other.
It is inherently desirable to produce a product having both acceptable efficacy and acceptable aesthetic attributes. Consequently, when making changes to a formulation in order to improve or optimise the product in one regard, be it sensory properties or efficacy, it is a practical desideratum that such changes do not impair to a significant extent the other properties of the product.
Structured hydrophylic emulsion antiperspirant formulations have been described in a series of recently published PCT applications to Unilever plc et al, including particularly WO00/61079, WO00/61081, WO00/61094 and WO00/61096. In particular, these specifications gave descriptions of various constituents which can be employed in antiperspirant emulsions, including antiperspirant actives, structurants, emulsifiers, constituents of the hydrophylic and non-hydrophylic phases and how to make such emulsions.
Various generalised ranges are given for the components of the structured antiperspirant emulsions and many different structured antiperspirant emulsion formulations are described in the aforementioned Unilever specifications, but there is no direct teaching therein as to which combinations of ingredient proportions have superior and which have inferior efficacy and/or sensory properties. Consequently, the problem remains as to how to select structured antiperspirant emulsions which can provide an improved balance of antiperspirant activity and sensory properties. In other words, the problem remains of how to avoid or ameliorate impairment of sensory properties when improving or maintaining efficacy at the same time as avoiding or ameliorating impairment of efficacy when improving or maintaining sensory properties.
A structured antiperspirant emulsion comprising an hydrophilic phase containing an aluminium and/or zirconium astringent salt dispersed in a continuous oil phase comprising a water-immiscible oil that is structured by a structurant in which the hydrophilic phase comprises from 25 to 55% by weight of the emulsion;
the hydrophilic phase contains a polyhydric alcohol in an amount of from 0 to not more than 15% by weight of the emulsion;
the emulsifier comprises an alkyl dimethicone copolyol;
the weight ratio of the hydrophilic phase to the emulsifier is selected in the range of from at least 60:1 the structurant comprises an acylated sugar and the water-immiscible oil and the structurant are present in a weight ratio of from 1.5:1 to 8.5:1.
By employing, in combination, a proportion of hydrophylic phase that is below a ceiling and containing none or not more than a ceiling proportion of a polyhydric alcohol; selecting a particular class of emulsifier in a specified weight ratio range to the hydrophilic phase, from at least or above a threshold ratio of hydrophylic phase to emulsifier of 60:1; and a specified class of structurants in a selected range of weight ratios to the oil phase which it structures, it is possible to attain, simultaneously, an improved combination of sensory and efficacy properties for the structured antiperspirant product.
The present invention relates to structured hydrophylic emulsions containing a specified range of proportions of hydrophylic phase, content of polyhydric alcohol, content of selected class of emulsifier, and content of specified class of structurant in a specified proportion within the emulsion, in order to attain a desired combination of sensory and efficacy properties.
Since the emulsifier constitutes the interface between the hydrophylic and oil phases, it is considered herein as a separate component and not within either phase for the purpose of calculating the weight proportion of either phase.
One of the sensory properties of an antiperspirant composition to which consumers pay particular attention is its stickiness. A number of consumers find it to be an unpleasant sensation if an applicator sticks to the skin during application of the antiperspirant formulation or if, subsequently, the skin sticks together.
Two factors have been identified for emulsions whose oil phase is structured with an acylated sugar. One factor comprises the proportion of polyhydric alcohol in the aqueous phase and the second factor comprises the proportion of the emulsion constituted by internal hydrophylic phase. It can be very desirable to incorporate polyhydric alcohol in the hydrophylic phase of an emulsion, for example as a means to adjust its refractive index or impart moisturising capability, but if too much of the polyhydric alcohol is present, the sensory properties of the emulsion can become progressively impaired. Likewise, although it can be desirable to employ a high internal phase volume, it has been found that this benefit would be achieved at the cost of relatively impaired sensory properties.
As indicated hereinabove, the proportion of internal phase in the instant invention has a ceiling of 55% by weight and the emulsion contains no more than 15% weight of polyhydric alcohol, which in fact will form part of the hydrophylic phase. By employing those two factors in combination, it is possible display good sensory properties, such as good absence of user-perceived stickiness.
However, it is recognised that the employment of a weight proportion of internal hydrophylic phase that is at the lower to middle parts of the practically possible range (maximum or high, commonly about 85%) of internal phase in an emulsion consequently limits the proportion of antiperspirant active which can be incorporated. Accordingly, the inventors sought ways in which the formulation could be controlled so as to improve or is optimise the delivery of the antiperspirant active to the eccrine sweat glands. What they found was that it was of considerable significant to control the weight proportion of the selected emulsifier to the hydrophylic phase. Surprisingly, too much of the selected class of emulsifier is disadvantageous, because it appears to interfere with the delivery of the antiperspirant active to the sweat glands. Accordingly, the instant invention employs a selected range of weight ratios of the emulsifier to the hydrophylic phase. Thirdly, the proportion of structurant is chosen within a specified range that takes into account the consequence of employing a comparatively low internal phase volume.
In many desirable embodiments the proportion of the hydrophylic phase is chosen in the range of from 30 to 50% by weight of the emulsion, often up to 45% by weight and particularly up to 40% by weight. A particularly desirable proportion comprises from 30 to 40% by weight.
The hydrophylic phase can comprise water as the sole hydrophilic liquid. However, additionally, the phase can comprise one or more polyhydric alcohols. Compared with solely employing water as the hydrophylic liquid, their substitution for a fraction of the water permits the refractive index of the phase to be increased, if that is so desired, such as to assist in the formation of a clear product by matching the refractive indexes of the internal and continuous phases.
Preferably, the weight proportion of the polyhydric alcohol is not more than 10% in the emulsion and in a number of beneficial embodiments is at least 1%. In a number of desirable embodiments, the weight ratio of water to polyhydric alcohol in the hydrophylic phase is in the range of from 1:1 to 30:1, and a preferred range comprises from 2:1 to 15:1. Expressed as a weight fraction of the hydrophylic phase, the polyhydric alcohol preferably comprises no more than about {fraction (2/7)}ths.
The polyhydric alcohol is desirably an aliphatic di or trihydric alcohol. Often it contains not more than 6 carbons, and in many instances is selected from propylene glycol, dipropylene glycol, glycerol, and the ether derived from propylene glycol and glycerol. Other polyhydric alcohols which can be contemplated comprise 1,2-hexane diol and 1,3 butane diol.
The emulsions according to the present invention described herein comprise the disperse hydrophobic phase mostly as a minor weight proportion or the remainder at no more than 55% of the emulsion in order for them to enjoy better sensory properties. It is highly desirable for the hydrophylic phase to contain the antiperspirant active salt at a comparatively high concentration. Preferably, the concentration of the antiperspirant active salt is at least 45% and particularly at least 50% by weight of the phase. In at least some preferred embodiments, it is at least 55% by weight. The concentration of the antiperspirant active is commonly less than 70% by weight and in many instances is not greater than 65% by weight.
It is particularly desirable to employ a comparatively high concentration of antiperspirant active in the disperse phase in conjunction with a comparatively low proportion of disperse phase in the emulsion. By a suitable choice of a high concentration of antiperspirant active in the hydrophylic phase and a suitably chosen proportion of hydrophylic phase in the emulsion, it is possible to obtain the benefits of having at least a desired or preferred proportion of antiperspirant active salt in the formulation which can enjoy superior sensory properties compared with formulations containing a similar proportion of antiperspirant salt, but with a higher proportion of disperse phase. For example, in some especially desirable emulsions, the formulations contain from 30 to 40% by weight of the hydrophylic phase, in which the antiperspirant active salt is present in a concentration selected in the range of from 55 to 65% by weight of that disperse phase.
Commonly, the proportion of antiperspirant active salt in many formulations that are preferred herein comprises at least 15% by weight of the emulsion and often up to 30% by weight. In advantageous emulsions herein, the proportion of the antiperspirant salt is at least 18% by weight, and in a number of particularly desirable embodiments, the emulsion contains from 18 to 20% and in others from 20 to 26% by weight of the aluminium and/or zirconium astringent salt.
It has been found that by varying the concentration of antiperspirant active in the hydrophylic phase, and by incorporating to the extent where necessary polyhydric aliphatic alcohol, it is possible to obtain an hydrophylic phase having a refractive index of at least 1.42 such as in the range of 1.42 to 1.45. In a number of embodiments, the refractive index is at least 1.44.
It is an advantage of many emulsions according to the present invention that by the formation of a disperse phase having the antiperspirant salt at a high concentration, it is possible to obtain an elevated refractive index value with the substitution of less polyhydric alcohol for water. Thus, when comparing hydrophylic phases having the same content of antiperspirant active, one being in accordance with the invention and one having a lower concentration of active in the phase, the same refractive index can be attained with less content of polyhydric alcohol. Accordingly, it is possible to bring together the advantages of refractive index adjustment with the benefits of having a lower disperse phase proportion and a lower polyhydric alcohol content.
The antiperspirant active employed herein is an aluminium and/or zirconium astringent salt. Preferred astringent salts include aluminium, zirconium and aluminium/zirconium halides and halohydrate salts, such as chlorohydrates.
Aluminium halohydrates are usually defined by the general formula Al2(OH)xQy.wH2O in which Q represents chlorine, bromine or iodine, x is variable from 2 to 5 and x+y=6 while wH2O represents a variable amount of hydration. Especially effective aluminium halohydrate salts, known as activated aluminium chlorohydrates, are described in EP-A-6739 (Unilever NV et al), the contents of which specification is incorporated herein by reference.
Zirconium actives can usually be represented by the empirical general formula: ZrO(OH)2nxe2x88x92nzBz.wH2O in which z is a variable in the range of from 0.9 to 2.0 so that the value 2nxe2x88x92nz is zero or positive, n is the valency of B, and B is selected from the group consisting of chloride, other halide, sulphamate, sulphate and mixtures thereof. Possible hydration to a variable extent is represented by wH20. Preferable is that B represents chloride and the variable z lies in the range from 1.5 to 1.87. In practice, such zirconium salts are usually not employed by themselves, but as a component of a combined aluminium and zirconium-based antiperspirant.
The above aluminium and zirconium salts may have coordinated and/or bound water in various quantities and/or may be present as polymeric species, mixtures or complexes. In particular, zirconium hydroxy salts often represent a range of salts having various amounts of the hydroxy group. Zirconium aluminium chlorohydrate may be particularly preferred.
Antiperspirant complexes based on the above-mentioned astringent aluminium and/or zirconium salts can be employed. The complex often employs a compound with a carboxylate group, and advantageously this is an amino acid. Examples of suitable amino acids include dl-tryptophan, dl-xcex2-phenylalanine, dl-valine, dl-methionine and xcex2-alanine, and preferably glycine which has the formula CH3CH(NH2)CO2H.
It is highly desirable to employ complexes of a combination of aluminium halohydrates and zirconium chlorohydrates together with amino acids such as glycine, which are disclosed in U.S. Pat. No. 3,792,068 (Luedders et al). Certain of those Al/Zr complexes are commonly called ZAG in the literature. ZAG actives generally contain aluminium, zirconium and chloride with an Al/Zr ratio in a range from 2 to 10, especially 2 to 6, an Al/Cl ratio from 2.1 to 0.9 and a variable amount of glycine. Actives of this preferred type are available from Westwood, from Summit and from Reheis.
In calculating the proportion of astringent antiperspirant salt in the emulsion or hydrophylic phase thereof, the weight of any water of hydration is normally excluded.
By suitably selecting the weight ratio of disperse phase to emulsifier, it is possible to make the most effective use of the antiperspirant salt, by increasing the extent of sweat reduction attainable at a given weight proportion of antiperspirant active in the emulsion.
Herein, the proportion of the emulsifier is selected in conjunction with the proportion of hydrophylic phase, and in the weight ratio range of the latter to the former of at or above the threshold value of 60:1. In many preferred embodiments, the weight ratio of the disperse phase to the emulsifier is at least 80:1. The ratio of hydrophylic phase to emulsifier is usually not greater than 480:1 and in many instances is up to 360:1, and in some instances up to 180:1. Especially desirable results have been obtained by selecting the ratio in the range of from 100:1 to 150:1, and in a number of advantageous embodiments, the weight ratio of the disperse phase to the emulsifier is selected in the range of from 105:1 to 140:1. In other advantageous formulations, very desirable results have been obtained employing the ratio in the range of from 180:1 to 360:1.
It is particularly advantageous to employ a selected high weight ratio of disperse phase to emulsifier in conjunction with a high concentration of antiperspirant active in that disperse phase, such as a concentration of at least 50% by weight.
As has been described hereinbefore, the instant invention avoids the employment of too much emulsifier, but of course employs an amount effective to form an emulsion. The minimum effective amount will vary, in accordance with the skilled man""s understanding, in conjunction with the emulsion-forming process and process conditions employed, such as the rate and extent of shear, and the particular constituents of the hydrophylic and hydrophobic phases. The proportion of emulsifier in the composition is often selected in the range up to 0.75% by weight and in many instances from 0.2 to 0.6% by weight of the composition and in many preferred embodiments is selected in the range of from 0.25 up to 0.4% by weight of the emulsion. In other embodiments, the weight proportion of emulsifier may be up to 0.2%, such as at least 0.1%, and particularly from 0.125% to 0.2%.
The aqueous emulsions according to the invention herein comprise an oil phase which is structured. The oil phase comprises at least one water-immiscible carrier liquid. It will generally be desired that this carrier is liquid (in the absence of structurant) at temperatures of 15xc2x0 C. and above. It may have some volatility but its vapour pressure will generally be less than 4 kPa (30 mm Hg) at 25xc2x0 C. so that the material can be referred to as an oil or mixture of oils. Preferably the carrier liquid comprises silicone oils or hydrocarbon oils or a mixture thereof.
It is preferred that the hydrophobic carrier material includes a volatile liquid silicone, i.e. liquid polyorganosiloxane. To class as xe2x80x9cvolatilexe2x80x9d such material should have a measurable vapour pressure at 20 or 25xc2x0 C. Typically the vapour pressure of a volatile silicone lies in a range from 1 or 10 Pa up to 2 kPa at 25xc2x0 C.
Volatile polyorganosiloxanes can be linear or cyclic or mixtures thereof. Preferred cyclic siloxanes include polydimethylcyclosiloxanes and particularly those containing from 3 to 9 silicon atoms and preferably not more than 7 silicon atoms and most preferably from 4 to 6 silicon atoms, otherwise often referred to as cyclomethicones, including especially pentacycloinethicones. Preferred linear siloxanes include polydimethylsiloxanes containing from 3 to 9 silicon atoms. The volatile siloxanes normally by themselves exhibit viscosities of below 10xe2x88x925 m2/sec (10 centistokes), and particularly above 10xe2x88x927 m2/sec (0.1 centistokes), the linear siloxanes normally exhibiting a viscosity of below 5xc3x9710xe2x88x926 m2/sec (5 centistokes). The volatile silicones can also comprise branched linear or cyclic siloxanes such as the aforementioned linear or cyclic siloxanes substituted by one or more pendant xe2x80x94Oxe2x80x94Si(CH3)3 groups. Examples of commercially available silicone oils include oils having grade designations 344, 345, 244, 245 and 246 from Dow Corning Corporation; Silicone 7207 and Silicone 7158 from Union Carbide Corporation; and SF1202 from General Electric.
The hydrophobic carrier employed in compositions herein can additionally comprise non-volatile silicone oils, which include polyalkyl siloxanes, polyalkylaryl siloxanes and polyethersiloxane copolymers. These can suitably be selected from dimethicone and dimethicone copolyols. Commercially available non-volatile silicone oils include Dow Corning 556 and Dow Corning 200 series.
The water-immiscible liquid carrier may contain from 0 to 100% by weight of one or more liquid silicones. Preferably, there is sufficient liquid silicone to provide at least 5% sometimes at least 10%, and on occasions at least 15%, by weight of the whole composition. If silicone oil is used, oil other than volatile silicone preferably comprises at least 20%, desirably at least 30% or at least 40% up to 100% of the weight of the water-immiscible carrier oils. In many instances, when a non-volatile silicone oil is present, its weight ratio to volatile silicone oil is chosen in the range of from 2:1 to 1:40.
Silicon-free hydrophobic oils can be used instead of, or more preferably in addition to liquid silicone oils. Silicon-free hydrophobic organic liquids which can be incorporated include liquid aliphatic hydrocarbons such as mineral oils or hydrogenated polyisobutene, often selected to exhibit a low viscosity. Further examples of liquid hydrocarbons are polydecene and paraffins and isoparaffins of at least 10 carbon atoms.
Other hydrophobic carriers are liquid aliphatic or aromatic esters or liquid aliphatic ethers, but these can be used as only part of the liquid carrier, in total desirably not above 20%, and possibly less than 10% by weight of the blend of water-immiscible oils.
Suitable aliphatic esters contain at least one long chain alkyl group, such as esters derived from C1 to C20 alkanols esterified with a C8 to C22 alkanoic acid or C6 to C10 alkanedioic acid. The alkanol and acid moieties or mixtures thereof are preferably selected such that they each have a melting point of below 20xc2x0 C. These esters include isopropyl myristate, lauryl myristate, isopropyl palmitate, diisopropyl sebacate and diisopropyl adipate.
Suitable liquid aromatic esters, preferably having a melting point of below 20xc2x0 C., include fatty alkyl benzoates. Examples of such esters include suitable C8 to C18 alkyl benzoates or mixtures thereof.
The liquid aliphatic ethers are desirably derived from at least one fatty alcohol, such as myristyl ether derivatives e.g. PPG-3 myristyl ether or lower alkyl ethers of polygylcols such as PPG-14 butyl ether Such liquid ethers preferably have a melting point of below 20xc2x0 C.
In many practical emulsions according to the present invention herein, the oil phase comprises a blend of oils including a volatile silicone oil, preferably in a weight proportion of not more than 90% of the blend and an oil other than volatile silicone oil, conveniently a non-volatile oil, suitably providing the balance of the blend. The proportion of non-volatile oil is preferably selected in the range of at least 10% up to 100%, and particularly from 15 to 70% or 85%. In many highly desirable emulsions according to the present invention, non-volatile oils constitute greater than 50% by weight of the blend of oils, such as from 55 to 70% or 70 to 85%. The non-volatile oils can be selected from the classes of oils described hereinabove, and particularly includes non-volatile hydrocarbon oils. A particularly desirable combination comprises pentacyclomethicone and polydecene. The presence of non-volatile oils is of assistance in increasing the refractive index of the oil phase compared with volatile silicone oils.
Herein, the emulsions employ a class of emulsifiers which comprises alkyl dimethicone copolymers, namely polyoxyalkylene modified dimethylpolysiloxanes. The polyoxyalkylene group is often a polyoxyethylene (POE) or polyoxypropylene (POP) or a copolymer of POE and POP. The copolymers often terminate in C1 to C22 alkyl groups, particularly C12 to C18. A suitable example comprises cetyl dimethicone copolymer (Abil EM90(trademark) from Th.Goldschmidt).
When greater than 10% by weight of the oil blend comprises other than a volatile silicone oil and the emulsifier comprises a dimethicone copolymer, it is advantageous to select the hydrophylic phase: emulsifier weight ratio in the range of from at least 100:1. In some embodiments, this is conveniently up to 140:1 or even higher, such as from 200:1 to 360:1.
It is especially desirable to employ a comparatively high weight ratio of disperse phase to emulsifier, such as at least 100:1 and particularly at least 105:1, for example in the ranges of ratios indicated hereinabove when the blend of oils in the oil phase contains at least 10% by weight of non-volatile oils, such as from 15% to 70% or 85%. It is furthermore especially desirable to employ such a comparatively high disperse phase:emulsifier weight ratio in conjunction with a comparatively low dispersed phase volume, such as up to 45% by weight of the emulsion and particular 30 to 40% by weight, and particularly when that disperse phase contains the antiperspirant salt at a comparatively high concentration within the phase, such as at least 50% and specially at least 55% by weight.
Herein the oil phase is structured using a structurant. Such a material comprises an acylated sugar (sometimes called an acylated saccharide) which is solid at ambient temperature, eg at 20xc2x0 C. and can form an homogeneous phase with the carrier oil or blend of oils on being heated to an elevated temperature and mixed together. On subsequent cooling, the structurant re-solidifies, thereby structuring the oil phase.
It is highly desirable herein to employ one or more structurants which are capable of forming a network of fibres. Such structurants are identified and described in greater detail in the above-identified WO00/61079. The fibres formed by such structurants commonly art thin (diameter less than 0.5 xcexcm, often less than 0.2 xcexcm) and are believed to have branches and/or interconnections.
An especially preferred class of saccharide derivative structurants comprises esters with a fatty acid, preferably of 6 to 13 carbon atoms especially 8 to 11 carbon atoms, including nonanoic acid in many instances. A suitable-saccharide derivative comprises acylated maltose. A preferred saccharide derivative comprises acylated cellobiose Preferably, the acylated saccharide is at least 70%, often at least 80% and in many embodiments is at least 90% esterified Herein, percent esterification indicates the average percentage of the total number of hydroxyl groups on the saccharide which have been esterified. For maltose and cellobiose, which each contain 8 hydroxyl groups prior to esterification, such preferred levels of acylation can be attained, conveniently, by a mixture of mainly octaesterification with some heptesterification. One especially preferred structurant comprises cellobiose nonanoate, i.e. cellobiose that has been esterified with nonanoic acid, preferably to at least 70 or 80%. Preferably, at least 75% and often at least 80% of the cellobiose nonanoate comprises the xcex1 anomer and the balance comprises the xcex2 anomer.
Although in a number of acylated sugars, the sugar So nucleus is acylated by a single fatty acid, e.g. nonanoic acid, in some others, the acyl group at for example the anomeric carbon in the sugar nucleus can be substituted by a different acyl group from that on the other carbon atoms in that nucleus. In such compounds containing ester groups derived from a plurality of different carboxylic acids around the sugar nucleus, such as around the cellobiose nucleus, at least a fraction of the ester residue at the anomeric carbon is different from ester residues of the remaining sugar nucleus carbons. The ester group at the anomeric carbon contains a saturated or unsaturated, linear or branched chain hydrocarbon residue containing from 1 to 31 carbon atoms optionally substituted or an aromatic hydrocarbon residue, optionally substituted or a cycloaliphatic hydrocarbon, optionally substituted of which at least a fraction is different from the ester substituents on non-anomeric carbons, eg different from an octyl residue from esterification with nonanoic acid. At least a major fraction of such acylated sugars which are acylated by two different carboxylic acids are in the form of the xcex2 anomer.
Methods for the synthesis of such acylated sugar structurants, where they are not commercially available, can be found in the afore-mentioned referenced specifications, for example WO00/61079, or by a suitable modification to such methods. Modifications can include the substitution of alternative sugars from which derivatives can be formed, and by reducing the ratio of acylating agent to sugar or reducing the reaction rate to allow the reaction to be halted with a fraction of the product not fully esterified and subsequently employing a different acylating agent to increase the extent of acylation.
The oil phase in the invention emulsions herein contain the water-immiscible oil or blend of oils and the structurant in the range of a weight ratios of from 1.5:1 to 8.5:1, particularly in a weight ratio of at least 2:1, and especially from 2.5:1. The weight ratio is often up to 7.5:1, and in many instances not higher than 6:1. It is highly desirable for the concentration of structurant in the emulsion to be at least 6%, and particularly at least 7.5% by weight of the emulsion, and in many instances is not more than 20% by weight of the emulsion.
The benefits of the instant invention are particularly evident when the structurant comprises or consists of a fibre-forming structurant, such as the aforementioned acylated sugars and especially an esterified cellobiose. However, if desired part, eg a minor fraction, of the fibre-forming structurant can be replaced by alternative structurants for an oil blend, such as a wax or a polymeric structurant.
Waxes herein are usually hydrocarbons, silicone polymers, esters of fatty acids or mixtures containing such compounds which are solid at ambient temperature and melt to a mobile liquid at a temperature above 30xc2x0 C. but generally below 95xc2x0 C. and preferably in a temperature range of 40xc2x0 C. to 90xc2x0 C. They can be produced from natural sources such as plants, eg candelilla, or animals, eg beeswax, or by processing oils, such as hydrogenated castor oil, or can comprise selected compounds therein, either extracted from such waxes or synthesised.
Polymeric structurants include polyamides or siloxane-polymers containing ester, urea, thiourea, or amide groups, as described in one or more of U.S. Pat. Nos. 5,603,925, 5,919,441 or U.S. Pat. No. 6,051,216. Further polymers which can be employed in the hydrophobic phase herein can comprise block copolymers of styrene with an alkylene containing up to 5 carbons, such as especially butadiene or ethylene/butylene. The block copolymers can comprise di-block or tri-block polymers such as those commonly abbreviated to SEB or SEBS copolymers. Commercially available block copolymers can be selected from polymers available under the tradenames Kraton and Transgel. Such copolymers can be employed in weight proportions of up to about 2% and in some instances conveniently from 0.2 to 0.8%.
The invention emulsions can comprise, if desired, one or more optional ingredients.
Optional ingredients in compositions of this invention can include deodorants, for example at a concentration of up to about 10% w/w. Suitable deodorant actives can comprise microbicides, including particularly bactericides, such as chlorinated aromatics, including triclosan, Triclorban(trademark), chlorhexidine and biguanide salts, the latter such as those available under the trade mark Cosmosil(trademark). Yet other highly effective deodorant actives can comprise strong chelators for iron such as diethylene-triaminepentaacetic acid (DTPA) and salts thereof which retard or suppress bacterial re-growth.
Other optional ingredients include wash-off agents, often present in an amount of up to 10% w/w to assist in the removal of the formulation from skin or clothing. Such wash-off agents are typically nonionic surfactants such as ethers containing a C8 to C22 alkyl moiety and a hydrophilic moiety which can comprise a polyoxyalkylene group (POE or POP) and/or a polyol. Other ingredients can include skin benefit agents such as glycerol, (already contemplated for incorporation in the hydrophylic phase) and allantoin or lipids, for example in an amount of up to 5%; soluble colorants; skin cooling agents such as menthol and menthol derivatives, often in an amount of up to 2%, all of these percentages being by weight of the emulsion. A commonly employed adjunct is a perfume, which is normally present at a concentration of from 0 to 4% and in many formulations from 0.25 to 2% by weight of the emulsion.
Further optional ingredients include particulate hydrophobic silica, such as in a weight proportion amount of up to 2%, conveniently from 0.2 to 0.7%. The silica is suspended in the oil phase. Desirably, the weight proportion of hydrophobic silica in the oil phase (including any carrier oil and structurant and any other water-immiscible material) is up to 2.5% and in a number of highly desirable formulations, the hydrophobic silica constitutes from 0.5 to 1.5% by weight of the oil phase. Such silica desirable has a low average particle size, often with no more than a minimal weight proportion (eg 0.1%) of above 40 microns diameter, such as is obtainable from fumed silica. The hydrophobic silica herein often has a specific surface area measured by a conventional BET method using nitrogen under standard conditions of at least 100 m2/g, such as from 100 to 350 m2/g, of which many are in the range of from 120 to 250 m2/g. Hydrophobic silicas within a wide range of bulk (free-flowing) density have been found to be desirable, such as from about 35 to about 220 g/l.
Commonly, the silica can be rendered hydrophobic at its surface in a conventional manner, such as by chemical reaction to form substituents of formula xe2x80x94OSiRaRbRc where Ra, Rb and Rc represent alkyl groups such as methyl. The measured carbon content (% by weight) of the hydrophobic silica is indicative of the proportion of the silica surface has been rendered hydrophobic, a larger % broadly corresponding to a higher proportion.
A low concentration, such as the concentrations indicated herein, of such hydrophobic silica has been found to improve the sensory properties of the emulsion stick. Furthermore, it has been found that the incorporation of such a small concentration of hydrophobic silica enables those formulations which demonstrate good light transmission in the absence of the silica to retain good transmission in its presence, thereby enabling the superior sensory property to be obtained without significant impairment of clarity.
It can be desirable to employ in the invention emulsions both a block copolymer, such as a SEB or SEBS copolymer together with a hydrophobic silica, each as and in their respective concentrations described hereinbefore. A convenient weight concentration in total of both such ingredients is from 0.5 to 2.0%.
The structured emulsions according to the present invention can be made by a process for the production of an antiperspirant composition comprising, concurrently or in any order, the steps of:
a) incorporating a structurant comprising an acylated sugar into a water-immiscible oil or blend of oils, forming an oil phase in a weight ratio of from 1.5:1 to
b) forming a hydrophylic phase comprising 25 to 55% by weight of the emulsion by dissolving an antiperspirant active in a hydrophylic solvent containing from 0 to not more than 15% by weight of polyhydric alcohol, based on the weight of the emulsion;
c) dispersing the hydrophylic phase within the oil phase in the presence of an alkyl dimethicone copolyol emulsifier in a weight ratio of the hydrophilic phase to the emulsifier in the range of from at least 60:1 and;
d) heating to an elevated temperature at which the structurant is soluble in the oil or blend of oils, followed by
e) introducing the mixture oil phase and disperse hydrophylic phase into a mould which preferably is a dispensing container, and then
f) cooling or permitting the mixture to cool to a temperature at which the oil phase is structured.
Optional ingredient can suitably be incorporated during the preparation of the relevant phase, or may be introduced into the mixture whilst it is mobile.
The emulsions herein are commonly made in the form of firm sticks. As such, they are capable of being dispensed using stick dispensers such as those described in WO0008970.
The invention formulations can be applied to skin in the conventional manner by wiping the sticks across the surface of the skin, and particularly in the axilla. This represents a non-therapeutic method of controlling perspiration and preventing or controlling the generation of body odour, otherwise commonly referred to as a cosmetic method.
Having provided a summary and preferred embodiments of the invention, specific embodiments thereof will now be particularised by way of example only. Variations thereto can be made by the skilled man in the light of the foregoing description.
In the following Examples, the ingredients employed in preparing the formulations were as follows:
For use in the Examples and comparisons herein, an acylated sugar structurant was prepared by a process substantially as described in Example 3 of WO00/01081. Cellobiose was acylated to produce an acylated ester in which at least 80% of the product had the xcex1 anomeric form, and had been esterified to at least 80% with nonanoate groups.
For use in certain of the Examples, concentrated aqueous solutions of the antiperspirant astringent salt were prepared in a preliminary step by introducing the particulate Al/Zr tetrachlorohydrex (ingredient 5) gradually with stirring into deionised water in a precalculated weight ratio thereto to achieve the desired concentration with gentle heating, optionally also in the presence of glycerol.
The Example Formulations and Comparison Formulations Were Made by the Following General Method:
In a suitably dimensioned flask, the cyclomethicone (volatile silicone oil) was mixed with the other organic oils (if any) and the cetyl dimethicone copolyol which functioned as an emulsifier, and the mixture was heated with gentle stirring to a temperature 5 to 10xc2x0 C. above the temperature at which the structurant had been found to dissolve (in an earlier test). The structurant, ie an esterified cellobiose, was then added and allowed to dissolve in the blend of oils with stirring whilst the temperature was maintained by heating.
The disperse phase (also referred to as internal phase) comprised the aluminium zirconium active dissolved in water or in a mixture containing the polyhydric alcohol. This disperse phase was pre-heated to the same temperature as the organic oils containing the esterified cellobiose and added slowly to them over a period of one minute while mixing with a Silverson mixer. After addition was complete, the formulation was mixed at higher speed for five minutes. Stirring speed was then reduced for a further one minute, after which the mixture was poured into stick barrels and allowed to cool undisturbed to ambient laboratory temperature.
In many of the Examples, the constituents of the continuous and disperse phases and their proportions were chosen not only to produce an emulsion having the desired sensory and efficacy properties, but also by taking into account their refractive indices, phases refractive index-matched to within 0.01 were obtained.
In the Examples, DPW indicates dispersed phase weight.

Abstract: i) the hydrophilic phase comprises 25 to 55%;

Abstract: ii) the hydrophilic phase contains 0 to 15% polyhydric alcohol;

Abstract: iii) the emulsifier comprises an alkyl dimethicone copolyol;

Abstract: iv) the weight ratio of the hydrophilic phase to the emulsifier is selected in the range of at least 60:1

Abstract: v) the structurant comprises an acylated sugar and

Abstract: vi) the water-immiscible oil and the structurant are present in a weight ratio of 1.5:1 to 8.5:1.

Unilever Home and Personal Care USA, division of Conopco, Inc.


Optical structures and methods for manufacturing the same includes, in one embodiment, a substrate and a plurality of two-sided optical components disposed along the substrate. Each component includes optical microstructures on each side. At least a portion of one side of at least some of the components is air-backed and the other side of the at least some of the components is substantially wetted-out by a material. Retroreflective optical structures, threads, or fibers and manufacturing methods for forming same are also provided. Optical structures are provided that include a plurality of microstructures enclosed within an outer layer that is formed from a single substrate. An apparatus and method are also provided for forming an optical structure comprising injecting a material into a mold to form the optical structure, forming the optical structure into a desired geometric shape, and sealing ends of the optical structure.

Retroreflective materials are employed for various safety and decorative purposes. Particularly, these materials are useful at nighttime when visibility is important under low light conditions. With perfect retroreflective materials, light rays are reflected essentially towards a light source in a substantially parallel path along an axis of retroreflectivity.
Many types of retroreflective material exist for various purposes. These retroreflective materials can be used as reflective tapes and patches for clothing, such as vests and belts. Also, retroreflective materials can be used on posts, barrels, traffic cone collars, highway signs, warning reflectors, etc. Retroreflective material can be comprised of arrays of randomly oriented micron diameter spheres or close packed cube-corner (prismatic) arrays.
Cube-corner or prismatic retroreflectors are described in U.S. Pat. No. 3,712,706, issued to Stamm on Jan. 23, 1973, the teachings of which are incorporated herein by reference. Generally, the prisms are made by forming a master negative die on a flat surface of a metal plate or other suitable material. To form the cube-corners, three series of V-shaped grooves, each series of grooves being parallel and equidistant to the other grooves in the same series, are inscribed in the flat plate such that the grooves intersect at 60 degrees. The die is then used to process the desired cube-corner array into a rigid flat plastic surface.
Further details concerning the structures and operation of cube-corner microprisms can be found in U.S. Pat. No. 3,684,348, issued to Rowland on Aug. 15, 1972, the teachings of which are incorporated herein by reference. A method for making retroreflective sheeting is also disclosed in U.S. Pat. No. 3,689,346, issued to Rowland on Sep. 5, 1972, the teachings of which are incorporated herein by reference. The disclosed method is for forming cube-corner microprisms in a cooperatively configured mold. The prisms are bonded to sheeting that is applied thereover to provide a composite structure in which the cube-corner formations project from one surface of the sheeting.