US20060159740A1

US20060159740A1 - Hydrophobic photosensitizer formulations for photodynamic therapy

Info

Publication number: US20060159740A1
Application number: US11/384,767
Authority: US
Inventors: Volker Albrecht; Alfred Fahr; Dietrich Scheglmann; Susanna Grafe; Wolfgang Neuberger
Original assignee: Ceramoptec Industries Inc
Current assignee: Ceramoptec Industries Inc
Priority date: 2003-08-26
Filing date: 2006-03-20
Publication date: 2006-07-20
Also published as: AU2004270119A1; EP2471512B1; DK1660041T3; JP5639014B2; KR20080110685A; EP2471512A3; JP4869929B2; CA2536977C; ES2403601T3; US20060088584A1; US20050048109A1; JP2007503436A; EP1660041B1; CN1842322B; KR100973064B1; PL1660041T3; WO2005023220A1; BRPI0413889A; RU2343904C2; PL2471512T3

Abstract

Pharmaceutical liposomal formulations for photodynamic therapy are presented, which are stable in storage as liquid formulations, comprise a hydrophobic photosensitizer and one or more synthetic phospholipids. The liposomal formulations provide therapeutically effective amounts of the photosensitizer for intravenous administration. The formed liposomes contain the hydrophobic photosensitizer within the lipid bilayer membrane. In the present formulation the size of the liposomal vehicles and their content of a therapeutically effective amount of the photosensitizing agent remain unchanged over storage times of a year or more, thus making the liquid formulations commercially viable. Being stable in the liquid state also makes them easy to store, and easier to use for doctors and patients. They can be prepared in a ‘factory setting’ delivered to practitioners in a liquid state and be available for use in PDT related treatments as called for by the practitioners' patient needs.

Description

REFERENCE TO RELATED CASE

This application is a continuation and divisional of co-pending U.S. patent application Ser. No. 10/648,168 filed on Aug. 26, 2003 by Volker Albrecht et al., inventors, entitled “Non-polar Photosensitizer formulations for PhotoDynamic Therapy”, and incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention concerns the preparation of stable liquid liposomal formulations for hydrophobic photosensitizers, which do not require freeze-drying and their use in therapy, particularly using intravenous injection.
2. Information Disclosure Statement
Liposomes are artificial vesicles composed of concentric lipid bilayers separated by water-compartments and have been extensively investigated as drug delivery vehicles. Due to their structure, chemical composition and colloidal size, all of which can be well controlled by preparation methods, liposomes exhibit several properties which may be useful in various applications. The most important properties are colloidal size, i.e. rather uniform particle size distributions in the range from 20 nm to 10 μm, and special membrane and surface characteristics.
Liposomes are used as carriers for drugs and antigens because they can serve several different purposes (Storm & Crommelin, Pharmaceutical Science & Technology Today, 1, 19-31 1998). Liposome encapsulated drugs are inaccessible to metabolizing enzymes. Conversely, body components (such as erythrocytes or tissues at the injection site) are not directly exposed to the full dose of the drug. The Duration of drug action can be prolonged by liposomes because of a slower release of the drug in the body. Liposomes possessing a direction potential, that means, targeting options change the distribution of the drug over the body. Cells use endocytosis or phagocytosis mechanism to take up liposomes into the cytosol. Furthermore liposomes can protect a drug against degradation (e.g. metabolic degradation). Although sometimes successful, liposomes have limitations. Liposomes not only deliver drugs to diseased tissue, but also rapidly enter the liver, spleen, kidneys and Reticuloendothelial Systems, and leak drugs while in circulation (Harris & Chess, Nature, March 2003, 2, 214-221).
Pegylation is an alternative method to overcome these deficiencies. First, pegylation maintains drug levels within the therapeutic window for longer time periods and provides the drug as a long-circulating moiety that gradually degrades into smaller, more active, and/or easier to clear fragments. Second, it enables long-circulating drug-containing micro particulates or large macromolecules to slowly accumulate in pathological sites with affected vasculature or receptor expression and improves or enhances drug delivery in those areas. Third, it can help to achieve a better targeting effect for those targeted drugs and drug carriers which are supposed to reach pathological areas with diminished blood flow or with a low concentration of a target antigen. The benefits of pegylation typically result in an increased stability (temperature, pH, solvent, etc.), a significantly reduced immunogenicity and antigenicity, a resistance to proteases, maintenance of catalytic activity, and improvements in solubility, among other features, and an increased liquid stability of the product and reduced agitation-induced aggregation.
In the pharmaceutical arts, liposomal formulations are being used more frequently as drug delivery systems. Freeze drying has been the most accepted means to provide long term stability for liposomal formulations. Generally liposomal formulation are not stable through the freeze-dry process unless amounts of saccharides or other material is associated with liposomes during freeze-dry procedure.
Photodynamic therapy (PDT) is one of the most promising new techniques being explored for use in a variety of medical applications and is known as a well-recognized treatment for the destruction of tumors (“Pharmaceutical development and medical applications of porphyrin-type macrocycles”, T. D. Mody, J. Porphyrins Phthalocyanines, 4, 362-367 2000). Another important application of PDT is the treatment of infectious diseases due to pathogenic micro organisms including dermal, dental, suppurative, respiratory, gastro enteric, genital and other infections.
A constant problem in the treatment of infectious disease is the lack of specificity of the agents used for the treatment of disease, which results in the patient gaining a new set of maladies from the therapy.
The use of PDT for the treatment of various types of disease is limited due to the inherent features of photosensitizers. These include their high cost, extended retention in the host organism, substantial skin photo toxicity, background toxicity, low solubility in physiological solutions (which reduces its usefulness for intravascular administration as it can provoke thromboembolic accidents), and low targeting effectiveness. These disadvantages lead to the administration of extremely high doses of a photosensitizer, which dramatically increase the possibility of accumulation of the photosensitizer in non-damaged tissues and the accompanying risk of affecting non-damaged sites.
One of the prospective approaches to increase the specificity of photosensitizers and the effectiveness of PDT is a conjugation of a photosensitizer with a ligand-vector, which specifically binds to receptors on the surface of a target cell. A number of natural and synthetic molecules recognized by target cells can be used as such vectors. This approach is now used in the design of new generations of photosensitizers for the treatment of tumors (“Porphyrin-based photosensitizers for use in photodynamic therapy” E. D. Sternberg, D. Dolphin, C. Brueckner, Tetrahedron, 54, 4151-4202 1998).
Another approach to increase tumor selectivity by targeting photosensitizers to tumor cells is using liposomes, e.g. transferrin-conjugated liposomes (Derycke & De Witte, Int. J. Oncology 20, 181-187, 2002). Because non-conjugated liposomes are often easily recognized and eliminated by the reticuloendothelial system, PEG-ylated liposomes were used (Woodle & Lasic, Sterically stabilized liposomes, Biochim Biophys Acta 1113, 171-199, 1992; Dass et al., Enhanced anticancer therapy mediated by specialized liposomes. J Pharm Pharmacol 49, 972-975, 1997).
Since the application of photodynamic therapy in the treatment of cancer and other diseases is increasing rapidly, there is also a bigger demand for new photosensitizer formulations. These new photosensitizer formulations need to be stable, easy to manufacture and to handle in many cases preferably in liquid form. Furthermore, it is desirable, more hydrophobic photosensitizers, to be able to be used as they often, are able to accumulate in tissue in an efficient and selective manner.

OBJECTIVES AND BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide an improved photosensitizer formulation for use in photodynamic therapy (PDT).
It is another object of the present invention to incorporate hydrophobic photosensitizers into a liposomal membrane.
It is further object of the present invention to formulate liquid liposomal formulations, for hydrophobic photosensitizer, which have commercially valuable shelf life and do not require freeze drying.
It is yet another object of the present invention to provide a photosensitizer formulation with improved pharmacokinetic properties.
It is still another object of the present invention to improve the transport of hydrophobic photosensitizers through the cell membrane and thus increasing the efficacy of PDT.
Briefly stated, the present invention provides pharmaceutical liposomal formulations for photodynamic therapy, which are stable in storage as liquid formulations, and that comprise a hydrophobic photosensitizer and one or more synthetic phospholipids. These liposomal formulations provide therapeutically effective amounts of the photosensitizer for intravenous administration. The formed liposomes contain the hydrophobic photosensitizer within the lipid bilayer membrane. In the present formulation the size of the liposomal vehicles and their content of a therapeutically effective amount of the photosensitizing agent remain unchanged over storage times of a year or more, thus making the liquid formulations commercially viable. Being stable in the liquid state also makes them easy to store, and easier to use for doctors and patients. They can be prepared in a ‘factory setting’ delivered to practitioners in a liquid state and be available for use in PDT related treatments as called for by the practitioners' patient needs.
The above and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a gel filtration curve of liposomal formulated mTHPC. Both, lipid components and mTHPC show the same distribution over all fractions collected.
FIG. 2 shows the temoporfin concentration in tumor, skin and skeletal muscle as percentage of injected dose per gram tissue, following intravenous injection of 2 μg liquid liposomal formulated temoporfin per mouse. Each time point shows the mean of four determinations.
FIG. 3 (3 a, 3 b, 3 c) Shows the photographs of whole tumor 24 hours after PDT, injected with liquid liposomal formulated mTHPC dosage 0.15, 0.05, 0.03 mg/kg and DLI 0.5, 3 and 6 hours.
FIG. 4 shows the histological assessment of tumor tissue 24 hours after PDT for three different liquid liposomal formulated mTHPC dosage (0.03, 0.05, 0.15 mg/kg) and three different DLI (0.5, 3, 6 hours).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A pharmaceutical liposomal formulation for photodynamic therapy comprising a hydrophobic photosensitizer and one or more synthetic phospholipids, which are stable in storage without requiring freeze-drying, is described below. The liposomal formulation provides therapeutically effective amounts of the photosensitizer for intravenous administration. The formed liposomes contain the hydrophobic photosensitizer within the membrane and are useful for targeting of a hydrophobic photosensitizer at cancerous and other hyperproliferative cells.
Preferable synthetic phospholipids are phosphatidyl cholines such as dipalmitoyl phosphatidyl choline (DPPC), Distearoyl phosphatidyl choline (DSPC), dimyristoyl phosphatidyl choline (DMPC), and phosphatidyl glycerol such as dipalmitoyl phosphatidyl glycerol (DPPG), Distearoyl phosphatidyl glycerol (DSPG) and dimyristoyl phosphatidyl glycerol (DMPG).
The hydrophobic photosensitizers in this invention include the known chlorin and bacteriochlorin compounds that have light absorption maxima in the range of 640-780 nanometers. The formed liposomes contain the hydrophobic photosensitizer within the membrane and are useful for targeting of hydrophobic photosensitizer.
In this present invention the size of the liposomal vehicle as well as the membrane content of the photosensitizing compound is well preserved without freeze drying or adding saccharides.
The photosensitizing formulations are useful to target the hydrophobic photosensitizer molecule to the unwanted cells or tissues or other undesirable materials and, after irradiation with an appropriated light source, to damage the target. The photosensitizing formulations are also useful to monitor unwanted cells or tissues or other undesirable materials by using fluorescence imaging methods without or with limited photochemical activation of the photosensitizer.
Especially the liposomal formulation of the invention is useful to transport hydrophobic photosensitizers. Hydrophobic substances are integrated within the membrane of the vehicles, thereby creating a structure that opens up easier, freeing the photosensitizer for action directly to the cell membrane. This mechanism delivers the photosensitizer directly to the cellular membrane system, one preferred place of action. Thus the photosensitizer, being effectively activated by illumination with an appropriate external light source, can irreversibly damage unwanted cells, tissues or other structures.
Conventionally constructed liposomal formulations are used to transport several compounds trapped into the luminal part of the vehicle. The present invention focuses on the lipid bilayer as transport compartments. In this way, by residing photosensitizer within the membrane, photosensitizing agents are effectively targeted to their place of action but the luminal part inside the liposome particle stays free for the inclusion of other substances, including drugs that may have a beneficial effect on the therapy. The liquid liposomal formulation of mTHPC in this invention does not need to be freeze dried to have a stable commercially viable shelf life, unlike many conventional commercial liposomal formulations.

Example 1

Preparation of Liposomes Containing m-THPC
mTHPC (Temoporfin) was synthesized as described in U.S. Pat. Nos. 4,992,257 and 5,162,519, incorporated herein by reference.
Liposomes were prepared according to the following general procedure:
Hydrophobic photosensitizer, and the phospholipids are dissolved in chloroform/methanol. The solution is then dried under vacuum using a rotary evaporator until the chloroform/methanol mixture is not detectable by gas chromatography anymore. Water for injection is added to rehydrate the lipid film at a temperature of 50° C. for at least 2 hours. The mixture is then passed through a homogenizer filter system using a final pore size of 100 nanometer. The filtrate is collected, filled into vials and stored for further use. This liquid liposomal formulation of mTHPC can be administered using physiologically compatible solutions to have an isoosmolaric solution to reduce pain during injection.
In one of the embodiments of this invention liposomes are prepared in t-butanol solution wherein the hydrophobic photosensitizer and synthetic phospholipids are dissolved in t-butanol at 50° C. This solution is then cooled to below room temperature where t-butanol crystallizes out as its crystallization temperature is about 20° C. The powder is dispersed in water and passed through a homogenizer and suitable filter. The filtrate is collected and stored in vials for further use. This liquid liposomal formulation can be used with suitable physiologically compatible solutions or simply in an aqueous solution.
This liquid liposomal formulation of mTHPC without further processing, retains its size and stability without affecting the mTHPC content carried in the bilayer member of the liposome. The physical and chemical stability data given below shows this formulation to have a very commercially valuable, stable shelf life.

Using the foregoing procedure, several different preparations of the liposomal formulation were prepared as follows:



	Ingredient	Amount % w/v

	mTHPC	0.05 to 0.15
	Dipalmitoyl Phosphatidyl Choline	0.5 to 2.0
	Dipalmitoyl Phosphatidyl Glycerol	0.05 to 0.2
	Water for Injection	as required to achieve desired
		concentrations above

All were found to function well in their use according to the present invention. In the above formulations each of the phospholipids, dipalmitoyl phosphatidyl choline and dipalmitoyl phosphatidyl glycerol, are synthetically prepared, and are not isolatable from natural sources.

Example 2

Physical and Chemical Stability of Liposomal m-THPC
The physical stability of the liposomal formulations was measured by monitoring the particle size distribution by photon correlations spectroscopy.

Stability of liposomal mTHPC

Storage Conditions Mean Particle Size distribution (nm)

Initial 166

23° C. - 1 Month 177

23° C. - 4 Month 167

Example 3

Another set of specific experiments repeated for separate batches of the m-THPC were followed for a longer period of time are given below. Storage conditions were 25° C. and 60% RH.

Ingredient Amount mg/ml

mTHPC 0.6

Dipalmitoyl Phosphatidyl Choline 14.7

Dipalmitoyl Phosphatidyl Glycerol 1.36

Physiological solution for Injection as required for achieving desired concentrations above
The above formulation was evaluated for particle size, zeta potential and retention of active ingredient at the intervals below.

Initial 1 month 3 months 6 months 9 months

Particle size 122 122 110 110 123

Zeta-potential −64.42 −66.78 −63.60 −56.33 −49.23

m-THPC content 88.3 85.6 88.7 88.1 86.7

Example 4

Localization of mTHPC Within the Liposomal Bilayer of the Formulation
Gel filtration of liposomal formulation performed on Sephadex G50 columns. As shown in FIG. 1, lipids and mTHPC show the same distribution over all fractions indicating a physically interaction of both components i.e. integration of mTHPC into the membrane bilayer. The data related to the FIG. 1 is tabulated in table 1 and 2.

Example 5

Pharmacokinetic Studies for Liquid Liposomal Formulation of mTHPC in NMRI nu/nu Mice Bearing a HT29 Tumor.
HT29 human colorectal carcinoma cells are used.
Six to eight weeks old adult female athymic NMRI nu/nu mice (Harlan Winkelmann GmbH, Germany) weighting 22-24 g were inoculated subcutaneously in the left hind thigh with 0.1 ml of 8×10⁷HT29 human colorectal carcinoma cells/ml in 5% glucose. Two to three weeks later, as the tumor reached a surface diameter of 7-8 mm, and a thickness of 2-3 mm in height, 50 μL liposomal formulation of mTHPC (0.04 mg/ml) were injected into lateral tail vein.
Following the drug injection animals were held under normal animal house lighting conditions (average illumination of 200 Lux, maximum 600 Lux of fluorescent tube light, with no exposure to sunlight or daylight. No photo-reaction were observed during this period.
At a selected time points (0, 1, 2, 3, 4, 5, 6 and 8 hours) post injection, 4 mice at each time point were anesthetized and sacrificed and tissue samples of tumor, skin and skeletal muscle were weighted and stored at −70° C. Briefly, tissue samples were thawed and held on ice. All tissue samples were reduced to small pieces by cutting with a scalpel, weighed and freeze dried (Christ Freeze drying system Alpha 1-4 LSC). The resulting powdered tissue was weighed and approx. 10-20 mg was transferred to a 2.0 ml reaction tube. Then 1.5 ml of methanol:DMSO (3:5, v:v) was added followed by immediate mixing for three times five seconds using a vortex mixer (Merck Eurolab, MELB 1719) operating at 2,400 rpm and then incubated at 60° C. while continuously shaking for at least 12 hours. All samples were then spun at 16,000 g in a centrifuge (Microfuge, Heraeus, Germany) for five minutes. 1 ml of each supernatant was transferred to a HPLC vial and analyzed by HPLC. The fluorescence detector was set at 410 nm for excitation and 653 nm for emission. The tissue concentration of mTHPC was calculated from a calibration curve constructed by plotting the peak height values of mTHPC standard solutions versus their concentrations.
Results of this study show no adverse effects immediately after injecting liquid liposomal formulation of mTHPC. The subjective quality of each injection was recorded, since the mouse tail vein is quite small and the injection was not always successful. Examination of these data shows that perfect injections were achieved in about 95% of cases with liquid liposomal formulation of mTHPC. In those cases in which the injection was not successful, the animal was excluded from the experiment. In case there were indications of slight extravasation of the drug or slight leakage from the puncture whole, the animal was not excluded, but marked as having received an injection which was not perfect.
The results demonstrate that a liquid liposomal Temoporfin formulation of the present invention indeed shows faster pharmacokinetics than a conventional Temoporfin solution. The highest photosensitizer concentration in the tumour was already obtained 6 hours (FIG. 2) after injection whereas the highest levels in skin were reached within 2 hours after injection. The drug concentration in skeletal muscle peaked 5 hours after injection. In comparison to Temoporfin solution the tumour to skeletal muscle ratio is 2 times higher for liquid liposomal formulation of temoporfin. To summarize these results, we can conclude that a photodynamic therapy in this murine model will be feasible already after 6-7 hours post injection. This would mean that the Drug Light Interval (DLI) is reduced when compared to the conventional mTHPC solution formulation thus accelerating PDT in a clinical setting, making this form of cancer treatment even more comfortable and practicable, because injection and irradiation can take place on a single day.
Liquid liposomal formulation of mTHPC, does not contain any organic solvent and can as aqueous colloidal formulation be diluted with aqueous media like blood without precipitation. Common adverse reactions of conventional temoporfin in an ethanol/propylene glycol mixture) like pain at the site of injection and other site reaction was not observed in the liquid liposomal formulation of temoporfin.

Example 6

Antitumor Activity of Liposomal m-THPC
a. Drug Dose
Three different dosages (0.15, 0.05, 0.03 mg/kg body weight) of liquid liposomal formulation of mTHPC is used.
b. Cell Line
HT29, a metastasizing human colorectal tumor cell line was used. Cells were maintained as a monolayer culture in Dulbecco's modified Eagle medium (DMEM) completed with 10% heat-inactivated fetal calf serum, 100 μg/ml streptomycin, 100 i.U./ml penicillin, at 37° C., in 95% air and 5% CO₂.
c. Tumour Model
Six week old athymic female mice (NMRI, nu/nu) were inoculated subcutaneously into the right hind foot with 8×10⁶HT29 cells. 10 days later, as the tumour had reached a diameter of approx. 10 mm; the test substance was injected intravenously. Unless indicated otherwise, 4 mice per dose and per Drug-Light-Interval (DLI) were used.
d. Photodynamic Treatment
Drug-light interval (DLI) of 0.5 h, 3 h, and 6 h was used. Each animal was photo-irradiated at 652 nm with 10 J/cm²at 100 mW/cm²using a diode laser.
e. Evaluation of PDT Effect
The tumor tissue necrosis after PDT treatment is evaluated by histological methods using Hematoxylin Eosin stain (HE); Periodic Schiff stains (PAS).
Twenty four hours later the mice were sacrificed, tumors were ablated and photographed for different drug dose and drug light interval as shown in the FIG. 3
Results

a. Drug light interval (DLI)=0.5 h, No PDT effect was observed at any of the liquid liposomal mTHPC formulation concentrations used in the experiments (FIG. 3 a)
b. Drug light interval (DLI)=3 h. No PDT effects were observed at lower liquid liposomal formulations for mTHPC used in the experiment. But slight red coloring of the tumor tissue were observed for drug dosage of 0.15 mg/kg. (FIG. 3 b)
c. Drug Light Interval (DLI)=6 h, Distinct necrosis was noticed in the tumor of mice treated with 0.15 mg/kg of liquid liposomal formulation of mTHPC. While lower dosage of the formulation did not show any necrosis. (FIG. 3 c)

Histological evaluation (FIG. 4) of dissected tumour tissue 24 hrs after PDT indicates only slight necrosis for lower doses of liquid liposomal formulation of mTHPC. The necrosis was similar for all analyzed drug light intervals (DLI). Only a dose of 0.15 mg/kg of liquid liposomal formulated mTHPC used showed an increasing level of necrosis for 3 and 6 hours of DLI. FIG. 4 clearly shows the necrosis in the form of scores. Score 1 means no necrosis reported, score 2—small necrotic areas reported, score 3—indicates larger necrotic areas and score 4 meant distinct necrosis.

Conclusion

In the present experiment three different doses of liquid liposomal formulation of mTHPC and three different drug light intervals were tested. Effective necrosis was observed 24 hours after PDT macroscopically for a dosage of 0.15 mg/kg and DLI of 6 hour. A DLI of 3 hours and a dosage of 0.15 mg/kg caused already a slight redness 24 hours after PDT. With all the other dosage and DLI tested no effect was seen.
The histological assessment correlates with the macroscopic observation describes above. Only a dose of 0.15 mg/kg of liquid liposomal formulation of mTHPC generated a large necrosis using a DLI of 3 and 6 hours.
This observation correlates well with pharmacokinetic data obtained in other studies. Liquid liposomal formulation of temoporfin is characterized by faster pharmacokinetics than conventional temoporfin solution. Maximum concentration of mTHPC in tumor tissue was achieved 6 hours after intravenous administration of liquid liposomal formulation of temoporfin, when compared to the convention temoporfin solution which takes 24 hours to reach maximum tumour concentration.
Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

1. A pharmaceutical liposomal formulation for photodynamic therapy, which is stable without freeze-drying, comprising:

a liposomal bilayer which consists substantially of two or more synthetic phospholipids; and,

a therapeutically effective amount of a hydrophobic photosensitizer, contained within said liposomal bilayer.

2. The liposomal formulation according to claim 1, wherein said synthetic phospholipids are selected from the group consisting of dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, dipalmitoyl phosphatidyl glycerol, distearoyl phosphatidyl glycerol. and combinations of these materials.

3. The liposomal formulation according to claim 1, wherein said synthetic phospholipids are a combination of dipalmitoyl phosphatidyl choline and dipalmitoyl phosphatidyl glycerol.

4. The liposomal formulation according to claim 3, wherein the weight ratio of said phosphatidyl choline to said phosphatidyl glycerol is about 10:1.

5. The liposomal formulation according to claim 1, wherein said hydrophobic photosensitizer is selected from the group consisting of pheophorbide and dihydro and tetrahydro porphyrin compounds.

6. The liposomal formulation according to claim 5, wherein said hydrophobic photosensitizer is temoporfin.

7. The liposomal formulation according to claim 1, wherein said therapeutically effective amount of the photosensitizer is from 0.0001 to 0.15 percent w/v.