CN1964734A - Enzyme therapy for retinitis pigmentosa and related pharmaceutical composition in kit form - Google Patents

Abstract

A kit for the treatment of retinitis pigmentosa containing the coenzymes reduced Nicotinamide Adenine Dinucleotide (NADH), reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and reduced glutathione in aliquot parts and interactive quantities suitable for administering said enzymes according to a predetermined time sequence.

Description

Enzyme therapy for retinitis pigmentosa and related pharmaceutical composition in kit form

technical field

The present invention relates to an enzyme therapy of retinitis pigmentosa and a pharmaceutical composition in the form of a kit useful for this therapy.

Background technique

Retinitis pigmentosa is a disease of the retina that presents many different pathologies: it can lead to a restricted field of vision and increasing difficulty adapting to darkness and the penumbra when it affects the peripheral zone of the retina, which has a large proportion of rod cells and whose Provides possible vision in the penumbra and a sensation of movement in the lateral zone, or when the cones are modulated can result in loss of central vision. The rate of progression of the disease varies from patient to patient. Usually, retinitis pigmentosa manifests itself in adolescence, but it can also appear in children and manifest in a subtle manner.

The cause of the disease is as yet unknown and therefore no therapy exists for people suffering from this disease. The only definitive information available to specialists concerns the genetic origin of retinitis pigmentosa, which is inherited in part through generations, according to mechanisms known to geneticists. Most forms of retinitis pigmentosa are inherited and three forms of inheritance have been identified to date: autosomal dominant, autosomal recessive, and closely sex-linked (X-linked).

The main symptoms of the disease are blurred vision and night blindness, ie difficulty seeing when lighting conditions are poor, and problems adjusting from good light to dark environments or vice versa. This phenomenon is due to the fact that, at least in most cases, the onset of the early onset of the disease is concentrated in the rod cells. Other typical symptoms are a reaction to excessively bright light (glare), progressive narrowing of the visual field in the form of difficulty perceiving objects located on the sides or tripping over steps or other low obstacles, and eventually total blindness.

The course of the disease is of extremely variable duration, but is often gradual and eventually disabling. The previously described symptoms worsened in most cases and the field of vision became increasingly restricted and eventually completely blind. Other disorders in which it is prone are glare, color blindness, and certain forms of cataracts. The end result in most cases is unfortunately complete blindness.

To diagnose the disorder, tests such as fundus examination, visual field examination, electroretinogram, fluorescein angiography, and visual acuity tests are usually relied on:

-Fundus examination aims to assess the condition of the retina and to look for the presence of characteristic pigmented macules on the retinal surface, which in this disease have a characteristic "osteoblast-like" appearance. Although they present the same symptoms, some rare forms of retinitis pigmentosa are not characterized by spots on the fundus;

- Examination of the visual field makes it possible to assess the sensitivity of the various parts of the retina to light stimuli. It is useful to have an objective record of the visual difficulties experienced by the patient;

- The electroretinogram (ERG) consists of recording the electrical activity of the retina in response to specific light stimuli, thus enabling a definitive assessment of the functionality of two different types of photoreceptors, namely cones and rods. The electroretinogram is a very important test for the diagnosis of retinitis pigmentosa because - even when the disease is in its initial stages - the resulting depiction is often very flat or completely absent;

- Fluorescein angiography is performed by intravenous injection of fluorescent substance and subsequent photography of the retina at different times. In fact, thanks to the blood circulation, the fluorescent substance reaches the retina, where it stains arteries, capillaries and veins and thus makes them visible, also reflecting the functional state of their walls;

- Vision examination allows assessment of visual acuity and consists of the patient reading letters of different sizes at a distance of three meters.

Although retinitis pigmentosa was identified and classified around the middle of the last century, there has been little concrete progress to date on the prospect of a possible cure or the equally important prospect of understanding what determines and regulates its progression. The most widely used method in international research at present is the genetic method, which seeks to identify the gene or genes responsible for the disease and allows subsequent intervention through modern genetic engineering techniques, namely, the transplantation method, which aims at the possible transplantation of retinal tissue Or at least the technique of transplanting healthy cells into the diseased retina, as well as immunological methods, which are used to test some theories that postulate that the disease is based on certain changes in the immune system.

WO 2004/030693 (same applicant) discloses a kit for the treatment of retinitis pigmentosa comprising glutathione peroxidase, aminoacylproline dipeptidase, glucose-6-phosphate dehydrogenase Enzymes and scheduled dosing sequence. With this treatment, although effective after a somewhat longer period of time, the patient responds rather slowly in terms of disappearance of the pigmented spots on the central retina and the vision remains blurred even long after the injection.

Contents of the invention

The object of the present invention is to provide a pharmaceutical composition in the form of a kit which allows a more effective treatment of retinitis pigmentosa than is possible with the therapy according to WO 2004/030693.

Another object of the present invention is to provide a method for the treatment of retinitis pigmentosa which results in a gradual restoration of visual acuity and visual field enlargement, image clarity and color vision faster than possible with the therapy disclosed in WO 2004/030693 and Finally, the normal electroretinogram was reconstructed.

According to the present invention, these objects are achieved by utilizing specific enzymes for incorporation in a pharmaceutical composition in the form of a kit for the treatment of retinitis pigmentosa by injection into the retrobulbar tissue, all as entitle as stated in Requirement 1.

More specifically, the enzymes used are reduced nicotinamide adenine dinucleotide (NADH), hereinafter referred to as enzyme A, reduced nicotinamide adenine dinucleotide phosphate (NADPH), hereinafter referred to as enzyme B, and reduced Glutathione, hereinafter referred to as Enzyme C, was administered according to the time sequence and form further specified below.

The enzymes used in the therapy of the present invention are commercially available in lyophilized form and dissolved in physiological solutions to make them useful in the therapy.

Each enzyme—in the form of an enzyme solution—was given by retrobulbar injection into each eye for three consecutive days, and said administration was repeated on the other two occasions, each time separated by a period of one month (for each enzyme ). In practice the method is as follows: the therapy begins with a single dose of Enzyme A injected into the retrobulbar tissue of each eye for three consecutive days, then the therapy is repeated in the second and third month; At the first, fifth, and sixth months, a single dose of Enzyme B was injected into the retrobulbar tissue of each eye for three consecutive days; at the seventh, eighth, and ninth months, a single dose of Enzyme B C was injected into the retrobulbar tissue of each eye for three consecutive days.

The doses of the different enzymes used per injection (for each eye) were as follows:

Enzyme A 0.25-0.35mg

Enzyme B 0.25-0.35mg

Enzyme C 0.9-1.1mg

The preferred doses of the different enzymes per injection are as follows:

Enzyme A 0.3mg

Enzyme B 0.3mg

Enzyme C 0.3mg

These doses remained the same for all patients, completely independent of the altered typology. In particular, the enzyme solution is prepared by the method described above in such a way that the amount can be contained in 0.4 ml of injection. For example, an enzyme solution suitable to provide a 0.4 ml injectable dose containing the preferred amount of enzyme described above is prepared as follows

Enzyme A:

A vial containing 10 mg of lyophilized enzyme was filled to 12.5 ml with physiological solution.

Enzyme B:

A vial containing 10 mg of lyophilized enzyme was filled to 12.5 ml with physiological solution.

Enzyme C:

A vial containing 25 mg of lyophilized enzyme was filled to 10 ml with physiological solution.

It will of course be clear that if one decides to vary the dosage of any one of the enzymes within the dosage ranges stated above, these ratios will also vary.

The enzymes are administered in the order described above and the injection cycles are continuous without interruption.

The kit of the present invention for the treatment of retinitis pigmentosa comprises equal parts and interactive amounts of the above-mentioned enzymes suitable for administration:

a) Enzyme A at a concentration between 0.25 and 0.35 mg in 0.4 ml of physiological solution for three consecutive days, once a month for three consecutive days in each eye;

b) Enzyme B at a concentration of 0.25 to 0.35 mg in 0.4 ml of physiological solution for three consecutive days starting from the next month after the last enzyme A administration, once a month for three months in each eye;

c) Enzyme C at a concentration of 0.9 to 1.1 mg in 0.4 ml of physiological solution for three consecutive days starting from the next month after the last enzyme B administration, once a month for three months in each eye;

In particular, the enzymes are contained in each kit in lyophilized form and in amounts sufficient for at least one complete series of administrations, each enzyme subdivided to contain sufficient for one three-month injection cycle, i.e. 18 injections, Or an aliquot of the amount for one daily administration, ie two injections, and optionally a suitable dose of a physiological solution constituting said aliquot. In particular, the different enzymes in each kit are subdivided into one or more aliquots, each comprising, in the preferred dosage forms described above, 0.30 mg to 5.4 mg of enzyme A, 0.3 mg to 5.4 mg of enzyme B, 1.0 mg to 18 mg of Enzyme C. There may also be three or more aliquots of physiological solution of 0.4 to 7.2 ml each.

A gradual improvement in visual acuity and field of vision, color vision and image clarity (resolution) was observed in patients treated with the present invention. Her electroretinogram gradually improved and was finally reconstructed. In particular, the patient's response was faster in terms of disappearance of the pigmented macule in the central retina compared to the therapy of WO 2004/030693. Contrary to what often happens with WO 2004/030693 therapy, patients were reported to have clearer vision and especially unblurred vision 24-48 hours after injection. The given therapy produced positive responses in all patients, albeit over different time periods. Follow-up examination after 2-3 years showed that the improvement obtained was permanent and did not bring any side effects of any nature.

The results of the therapeutic therapy of the present invention on 23 patients with retinitis pigmentosa are summarized in Table 1 below. Means and standard deviations of the following parameters related to the right and left eyes before and after the treatment are presented: visual acuity, visual field and electroretinogram.

Table 1

Visual acuity Field of view(*) Electroretinogram RE LE RE LE RE LE   Before treatment After treatment Mean S.D. Mean S.D. 3,7/103,1/107,9/102,9/10 4,0/103,1/107,6/102,7/10 4,95% 3,33% 51,3% 15,2% 4,74% 3,32% 47,7% 17,3% No or greatly reduced -increased-

(*) Corresponds to the mean value of the temporal, nasal, superior and inferior lines of vision (isoptera).

Statistical analysis of each parameter indicated that the difference between the values before and after treatment was statistically significant.

Experimental data confirm the hypothesis that retinitis pigmentosa may arise from an enzyme defect that alters the metabolism of the retina, not only regulating the visual process but also promoting the accumulation of pigment that is a characteristic feature of the disease.

Claims (10)

1. A kit for the treatment of retinitis pigmentosa comprising equal parts and interactive amounts of enzyme nicotinamide adenine dinucleotide (enzyme A), nicotinamide adenine dinucleotide phosphate ( Enzyme B) and reduced glutathione (enzyme C): a) Enzyme A at a concentration between 0.25 and 0.35 mg in 0.4 ml of physiological solution for three consecutive days, once a month for three months in each eye; b) Enzyme B at a concentration of 0.25 to 0.35 mg in 0.4 ml of physiological solution for three consecutive days starting from the next month after the last enzyme A administration, once a month for three months in each eye; c) Enzyme C at a concentration of 0.9 to 1.1 mg in 0.4 ml of physiological solution for three consecutive days, starting one month after the last enzyme B administration, once a month for three months in each eye. 2. The kit according to claim 1, wherein the concentration of enzyme A is 0.3 mg in 0.4 ml of physiological solution, the concentration of enzyme B is 0.3 mg in 0.4 ml of physiological solution and the concentration of enzyme C is 1.0 mg in 0.4 ml of physiological solution. 3. A kit according to claim 1 or claim 2, wherein said kit comprises, in lyophilized form, an amount sufficient for at least one series of administrations a) to c), subdivided for each enzyme comprising An aliquot of said enzyme sufficient for once trimester, monthly or daily administration and, alternatively, a suitable dose of physiological solution for constituting said aliquot. 4. Kit according to any one of claims 1 to 3, wherein said kit comprises said enzyme in lyophilized form subdivided into one or more aliquots, each aliquot comprising 0.30 to 5.4 mg of Enzyme A, 0.3 to 5.4 mg of enzyme B, 1.0 to 18 mg of enzyme C and optionally, 0.4 to 7.2 ml each of three or more aliquots of physiological solution. 5. Enzyme reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH) (enzyme B) and reduced glutathione (enzyme C) are used to prepare by Method of injection into retrobulbar tissues Use of a pharmaceutical composition in the form of a kit for the treatment of retinitis pigmentosa, said kit comprising aliquots and interacting amounts of said enzymes suitable for administration: a) Enzyme A at a concentration between 0.25 and 0.35 mg in 0.4 ml of physiological solution for three consecutive days, once a month for three months in each eye; b) Enzyme B at a concentration of 0.25 to 0.35 mg in 0.4 ml of physiological solution for three consecutive days starting from the next month after the last enzyme A administration, once a month for three months in each eye; c) Enzyme C at a concentration of 0.9 to 1.1 mg in 0.4 ml of physiological solution for three consecutive days, starting one month after the last enzyme B administration, once a month for three months in each eye. 6. The purposes of the enzyme according to claim 5, wherein the concentration of enzyme A is 0.3mg in 0.4ml physiological solution, the concentration of enzyme B is 0.3mg in 0.4ml physiological solution and the concentration of enzyme C is 1.0mg in 0.4ml physiological solution . 7. The use of an enzyme according to claim 5 or 6, wherein the kit comprises, in lyophilized form, an amount sufficient for at least one series of administrations a) to c), subdivided for each enzyme into an amount comprising sufficient An aliquot of the enzyme and, alternatively, an appropriate dose of a physiological solution to make up the aliquot is administered to each eye once for three months, once a month or daily. 8. Use of an enzyme according to any one of the preceding claims, wherein said kit comprises said enzyme in lyophilized form subdivided into one or more aliquots, each aliquot comprising 0.3 to 5.4 mg of Enzyme A, 0.3 to 5.4 mg of Enzyme B, 1.0 to 18 mg of Enzyme C and optionally, 0.4 to 7.2 ml each of three or more aliquots of physiological solution. 9. A method for the treatment of retinitis pigmentosa, which consists in administering by injecting the following enzymes into the retroocular tissues: a) Reduced nicotinamide adenine dinucleotide (NADH) (enzyme A) at a concentration between 0.25 and 0.35 mg in 0.4 ml of physiological solution for three consecutive days, once a month for three months in each eye; b) Reduced nicotinamide adenine dinucleotide phosphate (NADPH) at a concentration of 0.25 to 0.35 mg in 0.4 ml of physiological solution for three consecutive days starting one month after the last enzyme A dose (enzyme B ), once a month for three months in each eye; c) Reduced glutathione (enzyme C) at a concentration of 0.9 to 1.1 mg in 0.4 ml physiological solution for three consecutive days starting from the next month after the last enzyme B administration, once a month, per animal Eyes for three months. 10. The method according to claim 9, wherein the concentration of enzyme A is 0.3 mg in 0.4 ml of physiological solution, the concentration of enzyme B is 0.3 mg in 0.4 ml of physiological solution and the concentration of enzyme C is 1.0 mg in 0.4 ml of physiological solution.