Use of Tetracycline to Treat Lymphangioleiomyomatosis (LAM)

BACKGROUND

1. Field of the Invention The invention relates to the use of tetracyclines to treat lymphangioleiomyomatosis (LAM).

2. Background Information

Lymphangioleiomyomatosis (LAM) is an aggressive interstitial lung disease that is exclusively found in women. Slowly progressive dyspnea and increasing fatigue associated with recurrent events of spontaneous pneumothorax are the usual, albeit non-specific, clinical manifestations. Gross pathologic findings in this sporadically occurring disease include hyperinflated lungs with loss of lung parenchyma accompanied by the development of cysts diffusely distributed throughout the lung. At present, there is no effective treatment for LAM. Accordingly, there is a need for new, more effective, treatments. SUMMARY

The inventors have discovered that tetracylines are effective in treating the symptoms of LAM and slowing progression of the disease. Accordingly, it is an object of the invention to provide a method for treating a patient afflicted with LAM to relieve, reduce or alleviate the symptoms of LAM by administering an effective amount of tetracycline or a tetracycline derivative to the patient. The amount to be administered can be determined by the skilled medical practitioner without undue experimentation, and should be effective to reduce at least one symptom of LAM and/or slow progression of LAM. By "relieved, reduced or alleviated" is meant a measurable decrease in a symptom. Persons of skill in the art are familiar with means of measuring the levels of symptoms of LAM. In particular, exercise capacity and oxygen desaturation during exercise are considered to be suitable measurements. Pulmonary function tests should also be useful in this regard.

The tetracycline utilized in the present invention may be any of the readily available, pharmaceutically acceptable tetracyclines known in the medical art.

Included in this group of tetracyclines are those such as chlortetracycline, which is marketed under the tradename Acronize, Aureocina, Aureomycin, Biomitsin, Biomycin, and Chrysomykine; Demeclyeycline marketed as Ledermycin, Detravis, Meciclin, and Mexocine; Doxycyline marketed as Vibramycin, Vibramycin Hyclace,

Liomycin Vibradox, Panamycin, Titradox, Hydramycin, Tecacin; Lymecycline which is marketed as Armyl, Mucomycin, Tetramyl, Tetralysal; Methacycline which is marketed as Adriamicina, Cyclobiotic, Germicilclin, Globociclina, Megamycine, Pindex, Londomycin, Optimycin, Rondomycin; Minocycline which is marketed as Minocin, Klinomycin, Vectrin; Oxytetracycline which is marketed as Biostat,

Oxacycline, Oxatets, Oxydon, Oxymycin, Oxytan, Oxytetracid, Ryomycin, Stezazin, Tetraject, Terramycin, Tetramel, Tetran, Dendarcin, Dendarcin; Rolitetracycline marketed as Bristacin, Reverin, Superciclin, Syntetrex, Syntetrin, Synotodecin, Tetraverin, Transcycline, Velacicline, Velacycline; and Tetracycline marketed as Achromycin, Ambramycin, Cyclomycin, Polycycline, Tetrabon, and Tetracyn.

Furthermore, chemically modified tetracyclines (CMTs) which do not have antimicrobial activity may be used, such as 4-dedimethylaminotetracycline, 4- dedimethylamino-5-oxytetracycline, 4-dedimethylamino-7-chlorotetracycline, 4- hydroxy4-dedimethylaminotetracycline, 4-dedimethylamino- 12a-deoxytetracycline, 4-dedimethylamino- 11 -hydroxy- 12a-deoxytetracycline, 4-dedimethylamino-7- dimethylaminotetracycline, 6-dimethyl-6-deoxy-4-dedimethylaminotetracycline, 6-o- deoxy-5-hydroxy4-dedimethylaminotetracycline, 1 la-chlortetracycline, 12a- deoxytetracycline, incyclinide (Col-3, CollaGenex, Inc.), and 2-nitrilo analogs of tetracycline. The tetracycline compounds may be administered by any suitable route known to those of skill in the art, such as, for example, orally, intravenously, intramuscularly, intradermally, subcutaneously, rectally, via inhalation, etc. The preferred route is oral.

The amount of the tetracycline compound to be administered is expected to be in the range of 50-300 mg/day administered in single or divided doses. For doxycyline, one preferred dosage is 50-100 qd. Because the mechanism through which the effects of tetracycline exerts its effects on LAM is uncertain, it is not necessary that it be given in an antimicrobially effective dosage.

Symptoms expected to be relieved by the treatment include dyspnea, fatigue, spontaneous pneumothorax, and oxygen desaturation during exercise. Estimated loss of lung volume per year is approximately 80 to 100 cc/year. Any incremental improvement in lung volume would be considered a benefit of the treatment. Improvement in distance walked would also be a measureable endpoint. Because the presumed mechanism of action is to inhibit matrix metalloproteinases, proteins that

are responsible for the remodeling of the extracellular matrix in the lung, inhibition of lung degradation may be able to be evaluated by changes in the high resolution computed tomography of the lung. In a slowly progressive disease like LAM, unlike the aggressive and relatively rapid changes noted in cancer, slow destruction of lung tissue by invasive LAM cells becomes an important therapeutic target. It is the destruction of lung tissue that results in the respiratory failure in LAM. Although the therapeutic methods and results of the invention do not depend upon any particular theory, the inventors believe that doxycycline and related tetracyclines slow the destruction of the lung tissue. This application claims priority to U.S. provisional application no. 60/844,094, filed September 13, 2006, which is incorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Doxycycline decreases MMP-2 activity in LAM cells. A. Representative zymography of LAMD-SM cells treated with either "V" = vehicle or "D" = doxycycline. B. Data are graphed as the mean ± SEM derived from two experiments on cells isolated from two separate patients. The top band is pro-MMP-2 and the bottom band is active MMP-2. (Procedure described in Elliot SJ, Striker LJ, Stetler-Stevenson WG, Jacot TA, Striker GE. "Pentosan polysulfate decreases proliferation and net extracellular matrix production in mouse mesangial cells." J Am Soc Nephrol 10:62-68, 1999.)

Figure 2 shows resolution of acute respiratory failure in a woman with end-stage LAM after treatment with doxycycline. M: MMP-2 standard; C: Control female urine; Patient lanes 1-5. Lane 1: Two weeks of doxycycline treatment (100mg/ml); Lane 2: Three weeks of treatment; Lane 3: Four weeks of treatment at time of change to 35% FIO2; Lane 4: Four weeks and one day of treatment; Lane 5: Seven weeks of treatment. MMP-2/Creatinine = Ratio of MMP-2 to Cr levels in a urine sample. BD = Below the detection level of the MMP-2 ELISA. FIO2 indicates supplemental oxygen delivered by ventilator.

Figure 3 shows the experimental protocol for measuring the effects of doxycycline on exercise capacity.

Figure 4 shows the distance walked in a 6 minute walk test in patients treated with doxycycline. Each line represents an individual patient. Y is distance walked and x is timepoint (l=baseline, 2=4 weeks of treatment, 3=24 weeks of treatment).

Figure 5 shows SpO ₂ in patients treated with doxycycline. Each line represents an /individual patient. Y is oxygen saturation and x is timepoint, as described in Figure 4 legend.

DETAILED DESCRIPTION Definitions

By "MMP" is meant matrix metalloproteinase(s). Specific MMPs are identified by number, as customary in the art. (See, e.g. Nagase H., Visse,R.;

Murphy,G.

Structure and function of matrix metalloproteinases and TIMPs Cardiovasc Res. 2006 Feb 15;69(3):562-73). "Pro-MMP" indicates pre-cleaved form. "Active-MMP" indicates bioactive form.

"LAMD-SM cells" are cells that are isolated and propagated from the outgrowth of LAM lesions (see, e.g. Goncharova et al. J. Biol. Chem.. 277:30958-

30967, 2002). "Borg Scale" is Borg Rating of Perceived Exertion Scale, a patient rating of perceived exertion during activity.

Example 1

Doxycvcline increased pro-MMP-2. but decreased active MMP -2 in cell supernatant collected from LAMD-SM cells.

LAMD-SM cells were isolated as previously described (Goncharova et al. J. Biol.

Chem.. 277:30958-30967, 2002). The cells were treated with either vehicle (V) or doxycycline (D, lOug/ml) in the standard culture medium for 24 hours, supernatants collected and assayed for MMP-2 activity. Data are graphed as the mean ± SEM derived from two experiments on cells isolated from two separate patients.

Doxycycline decreased active MMP-2 in cell supernatant collected from LAMD-SM cells.

Example 2

Resolution of acute respiratory failure in a woman with end-stage LAM

A female patient with end stage LAM who was in acute respiratory failure was treated with doxycycline 100 mg qd. Urine samples were first normalized to creatinine levels and then submitted to gelatin zymography. A representative zymogram is depicted in Figure 2. Note that the activity of MMP-2 decreases as the treatment course progresses and oxygenation improves. Patient was weaned successfully from ventilatory support.

The improvement in this patient is consistent with the hypothesis that doxycycline slows or arrests progression of LAM.

Example 3

Increase in exercise capacity and SpO2 in LAM patients treated with doxycycline

Ten female patients between the ages of 30 and 68 who had been diagnosed with LAM (by biopsy in 7 subjects and HRCT [high resolution computed tomography] in 3 subjects) were treated with 50-100 mg qd doxycycline for 24 weeks, and tested using the protocol shown in Figure 3.

The results of the 6 minute walk test in 10 patients are shown in Figure 4. No patient showed deterioration of function over the treatment period, and a number showed moderate improvement compared to their historical control data. Oxygen usage for 8 patients over the treatment period is shown in Figure 5.

The results, summarized in Table 1 , showed that doxycyline improved exercise capacity as determined by 6 minute walk distance (6MWD) and Borg scales. In subjects who maintained a SpO ₂ greater than 83% during a 6 minute walk test at baseline, the lowest SpO ₂ improved in the Doxycycline group at 24 weeks. No significant correlation was found between urinary MMP-2 levels and improvement in 6-MWD. Some subjects never record a high level MMP-2 despite an improvement in their 6-MWD. The only subjects that show correlation are those with high baseline MMP-2. No subjects showed decline in exercise capacity or increased infections. Two subjects reported GI intolerance.

* Statistical analysis using ANOVA and Tukeys multiple comparison test. We conclude that Doxycycline improves exercise capacity and reduces oxygen desaturation in patients with LAM, and will accordingly be an effective treatment method.

All publications and patent applications cited herein are hereby incorporated by reference. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined not with reference to the description herein, but instead should be determined with reference to the appended claims along with their full scope of equivalents.