AU2004320491A1

AU2004320491A1 - Enzymatic treatment of retinitis pigmentosa and relevant pharmaceutical composition therefor in form of a kit

Info

Publication number: AU2004320491A1
Application number: AU2004320491A
Authority: AU
Inventors: Paola Ammannati; Roberto Giordani
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-06-08
Filing date: 2004-06-08
Publication date: 2005-12-22
Also published as: EP1753444A1; WO2005120544A1; RU2007100153A; BRPI0418858A; CA2567205A1; CN1964734A; JP2008501783A; IL179479A0

Description

WO2005/120544 PCT/IT2004/000331 -1 TITLE ENZYMATIC TREATMENT OF RETINITIS PIGMENTOSA AND RELEVANT PHARMACEUTICAL COMPOSITION THEREFOR IN FORM OF A KIT DESCRIPTION 5 Field of the invention The present invention concerns a treatment of retinitis pigmentosa by means of enzymes and a pharmaceutical composition in kit form that can be used for this treatment. 10 Description of the prior art Retinitis pigmentosa is a disease of the retina that presents many different pathological manifestations: it may bring about a restriction of the field of vision and create increasing difficulty in adapting to the dark and 15 to penumbra, when it affects the peripheral zones of the retina, which accommodate the greater part of the rod cells that render possible vision in penumbra and the perception of movement in the lateral zones, or may lead to loss of central vision when the cone cells are the ones 20 to undergo modification. The rate of progress of the illness varies from one subject to another. As a general rule, retinitis pigmentosa manifests itself in youth, but often also affects children and acts in an insidious manner. 25 The causes responsible for this infirmity are as yet unknown and, consequently, there does not exist any cure for those affected by it. The sole certain information at the disposal of experts regards the genetic origin of retinitis pigmentosa, which is in part transmitted by 30 heredity from generation to generation, following mechanisms that are known to geneticists. The greater part of the forms of retinitis pigmentosa are hereditary and WO2005/120544 PCT/IT2004/000331 - 2 three transmission modalities have so far been identified: dominantly autosomal, recessively autosomal and bound up with sex (X-linked). The principal symptoms of the illness are 5 crepuscular and nocturnal blindness, i.e. difficulty of seeing when the lighting conditions are poor, and problems of adaptation from well-lit to dark environments or vice versa. This phenomenon is due to the fact that, at least in the greater part of cases, attack in the early 10 development phases of the illness is concentrated on the rod cells. Other typical symptoms are the reaction to excessively strong light (dazzlement), a gradual narrowing of the visual field, which manifests itself in the form of difficulty in perceiving objects situated on either side 15 or stumbling over steps or other low obstacles, eventually arriving at complete blindness. The course of the illness is of extremely variable duration, but is always gradual and ultimately leads to disablement. In the greater part of cases the previously 20 described symptoms become aggravated, the visual field becomes more and more restricted and eventually closes completely. Other complaints tend to appear, among them dazzlement, incapacity of distinguishing colours and a particular form of cataract. In many cases the final 25 outcome is, unfortunately, absolute blindness. For the purpose of diagnosing the illness it is usual to rely on such tests as examination of the fundus of the eye, examination of the visual field, electroretinograms, fluorangiography, and visus 30 examination: - examination of the fundus of the eye aims at assessing the condition of the retina and to look for the WO2005/120544 PCT/IT2004/000331 - 3 presence of the characteristic pigment spots on the retinal surface, which in the illness assume a characteristic "osteoblast-like" appearance. Though they present the same symptoms, some rare forms of retinitis 5 pigmentosa are not however characterized by spots on the fundus of the eye; - examination of the visual field makes it possible to evaluate the sensitivity of the various parts of the retina to light stimuli. It will be useful to have 10 an objective documentation of the difficulty in visual perception experienced by the patient; - the electroretinogram (ERG) consists of recording the electrical activity of the retina in response to particular light stimuli, thus making possible 15 distinct valuations of the functionality of the two different types of photoreceptors (i.e. cone cells and rod cells). The electroretinogram is a very important examination for diagnosing retinitis pigmentosa, because even when the illness is in its initial stages - the 20 resulting trace is almost always either very flat or altogether absent; - fluorangiography is performed by means of the intravenous injection of a fluorescent substance and subsequent photography of the retina at different times. 25 Due to blood circulation, in fact, the fluorescent substance arrives at the retina, where it colours the arteries, the capillaries and the veins and thus renders them visible, as also the functional state of their walls; - visus examination permits a valuation of visual 30 acuity and consists of the patient reading letters of different sizes at a distance of three metres.

WO2005/120544 PCT/IT2004/000331 - 4 Although retinitis pigmentosa was identified and classified about midway through last century, very little concrete progress has so far been achieved either on the front of possible cures or on the equally important front 5 of understanding the causes that determine and regulate its course. The lines at present most widely followed by international research are the genetic approach, which seeks to identify the gene or genes responsible for the illness and thus permitting a subsequent intervention by 10 means of modern genetic engineering techniques, the transplant approach, which aims at perfecting a technique that would make possible the transplant of retinal tissue or, at least, the grafting of healthy cells into diseased retinas, and the immunological approach, which sets out to 15 verify some theories that assume the illness to be underlain by some alteration of the immunological system. W02004/030693, in the name of the same Applicants discloses a kit for the treatment of retinitis pigmentosa contaning the enzimes glutathione peroxidase, prolidase, 20 glucose-6.phosphate dehydrogenase and a predefined administration time sequence. With this treatment method, though effective after a somewhat long period of time, the patient's response is rather slow as regards the disappearance of the pigment spots on the macula area and 25 the vision keeps being dimmed even for a long time after the injection. The object of the present invention is to provide a pharmacological composition in kit form that will permit retinitis pigmentosa to be more efficiently treated than 30 possible with the treatment according to WO2004/030693. Another purpose of the present invention is to provide a method of treating retinitis pigmentosa that WO2005/120544 PCT/IT2004/000331 - 5 will permit a gradual recovery of visual acuity and enlargement of the field of vision, the sharpness of images and the perception of colours more quickly with respect to what is possible with the treatment disclosed 5 in WO2004/030693 and the reconstitution of a normal electroretinogram in the long run. According to the present invention, these aims are attained by means of the use of particular enzymes for incorporation in a pharmaceutical composition in kit form 10 to be employed for treating retinitis pigmentosa by means of injection into the retrobulbar tissue, all as specified in Claim 1. More particularly, the enzymes employed are reduced Nicotinamide adenine dinucleotide (NADH), hereinafter 15 referred to as Enzyme A, reduced Nicotinamide adenine dinucleotide phospate (NADPH), hereinafter referred to as Enzyme B, and reduced Glutathion, hereinafter referred to as Enzyme C, which are administered in accordance with a time sequence and modalities to be further specified 20 hereinbelow. The enzymes employed in the treatment in accordance with the present invention are commercially available in lyophilized form and are dissolved in physiological solution to render them available for the treatment. 25 Each enzyme - in the form of enzyme solution - is administered by means of retrobulbar injection into each eye for three consecutive days, repeating the administration on another two occasions, each separated from its predecessor by a period of one month (for each 30 enzyme). In practice the procedure is as follows: - one dose of Enzyme A is injected into the retrobulbar tissue of each eye for three consecutive days WO2005/120544 PCT/IT2004/000331 6 at the beginning of the treatment, the treatment being then repeated in the second and third month; - in the fourth, fifth and sixth month one dose of Enzyme B is injected into the retrobulbar tissue of each 5 eye for three consecutive days; - in the seventh, eighth and ninth month one dose of Enzyme C is injected into the retrobulbar tissue of each eye for three consecutive days. The doses of the various enzymes used at each 10 injection (for each eye) are as follows: Enzyme A 0.25 - 0.35 mg Enzyme B 0,25 - 0,35 mg Enzyme C 0,9 - 1,1 mg The preferred doses of the various enzymes at each 15 injection are as follows: Enzyme A 0,3 mg Enzyme B 0,3 mg Enzyme C 0,3 mg These doses remain the same for all patients, quite 20 independently of the typology of the alteration. In particular, the enzyme solutions are prepared in such a way that the quantities set out above may be contained within an injection of 0.4 ml. For example, the enzyme solutions suitable for providing injectable doses of 0.4 25 ml containing the preferred enzyme quantities as set out above are to be prepared as follows Enzyme A: Phial containing 10 mg of lyophilised enzyme, bring up to 12,5 ml with physiological solution. 30 Enzyme B: Phial containing 10 mg of lyophilised enzyme, bring up to 12,5 ml with physiological solution.

WO 2005/120544 PCT/IT2004/000331 - 7 Enzyme C: Phial containing 25 mg of lyophilised enzyme, bring up to 10 ml with physiological solution. Clearly naturally, these ratios will have to be 5 modified if it is decided to change the dose of any one of the enzymes within its dosing range as set out above. The enzymes are administered in the order in which they are stated above and the injection cycles are to be continued without interruption. 10 The kit used for treating retinitis pigmentosa in accordance with the present invention contains the aforementioned enzymes in aliquot parts and interactive quantities appropriate for administering: a) Enzyme A at a concentration comprised between 15 0,25 and 0,35 mg in 0.4 ml of physiological solution for three consecutive days, at monthly intervals, for three consecutive days and for each eye; b) Enzyme B, starting from the month following the last administration of Enzyme A, at a concentration of 20 0,25 to 0,35 mg in 0.4 ml of physiological solution for three consecutive days, at monthly intervals, for three months and for each eye; c) Enzyme C, starting from the month following the last administration of Enzyme B, at a concentration of 0,9 25 to 1,1 mg in 0.4 ml of physiological solution for three consecutive days, at monthly intervals, for three months and for each eye. In particular, the enzymes are contained in each kit in lyophilized form and in quantities sufficient for at 30 least one complete series of administrations, each enzyme being subdivided into aliquot parts containing a quantity sufficient for one three-month injection cycle, i.e.

WO2005/120544 PCT/IT2004/000331 - 8 eighteen injections, or for one daily administration, i.e. two injections, and optionally the appropriate doses of physiological solution for constituting said aliquot parts. In particular, in each kit the various enzymes are 5 subdivided into one or more aliquot parts, each of which contains, in the preferred dosing forms as set out above, from 0,30 mg to 5,4 mg of Enzyme A, from 0,3 mg to 5,4 mg of Enzyme B, from 1,0 mg to 18 mg of Enzyme C. Possibly there may also be present three or more aliquot parts of 10 physiological solution from 0.4 to 7.2 ml each. Patients subjected to the treatment in accordance with the invention found a gradual improvement of visual acuity and field of' vision, colour perception and image sharpness (definition). Their electroretinograms improved 15 little by little, eventually becoming reconstituted in the long run. In particular, in comparison to the treatment according to W02004/030693, the response of the patients was more rapid as regards the disappearance of the pigment spots of the macula area. In contrast to what often occurs 20 with the treatment according to W02004/030693, the patients reported a clearer vision and especially an undimmed vision already 24-48 hours after the injection. The administered treatment produced a positive response in all the patients, albeit over different periods of time. 25 Follow-up checks after 2-3 years showed that the obtained improvement was permanent and did not bring out side effects of any kind. In the following Table 1 the results of the therapeutic treatment according to the invention relevant 30 to 23 patients suffering from retinitis pigmentosa are summarized. The average value and standard deviation for the following parameters: visual acuity, field of vision WO2005/120544 PCT/IT2004/000331 9- and electroretinogram relevant to right eye and left eye, before and after the treatment are shown. Table 1 visual acuity field of electroretinogram RE LE vision (*) RE LE RE LE before average 3,7/10 4,0/10 4,95% 4,74% void or treatment very reduced S.D. 3,1/10 3,1/10 3,33% 3,32% after average 7,9/10 7,6/10 51,3% 47,7% increased treatment S.D. 2,9/10 2,7/10 15,2% 17,3% - (*) mean values relevant to temporal, nasal, upper 5 and lower isoptera. The statistical analysis of each parameter showed that the differences among the values, before and after the treatment, are statistically significant. The experimental data confirm the hypothesis that 10 retinitis pigmentosa could be due to an enzyme defect that alters the metabolism of the retina, modifying not only the vision process, but facilitating also the accumulation of pigments, the characteristic feature of the illness. 15

Claims

1. A pharmaceutical kit for the treatment of retinitis pigmentosa containing the enzymes Nicotinamide adenine dinucleotide (Enzyme A), Nicotinamide adenine dinucleotide 5 phosphate (Enzyme B), and reduced glutathione (Enzyme C) in aliquot parts and interactive quantities appropriate for administering: a) Enzyme A at a concentration comprised between 0,25 and 0,35 mg in 0,4 ml of physiological 10 solution for three consecutive days, at monthly intervals, for three months and for each eye; b) Enzyme B, starting from the month following the last administration of Enzyme A, at a concentration of 0,25 to 0,35 mg in 0,4 ml of 15 physiological solution for three consecutive days, at monthly intervals, for three months and for each eye; c) Enzyme C, starting from the month following the last administration of Enzyme B, at a 20 concentration of 0,9 to 1,1 mg in 0,4 ml of physiological solution for three consecutive days, at monthly intervals, for three months and for each eye.

2. A kit in accordance with claim 1, wherein the 25 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. 30

3. A kit in accordance with claim 1 or claim 2, wherein said kit comprises said enzymes in lyophilised form, in quantities sufficient for at least one series of WO2005/120544 PCT/IT2004/000331 - 11 administrations of from a) to c), subdivided into aliquot parts containing - for each enzyme - a quantity of enzyme sufficient for one three-months, monthly or daily administration and, optionally, the appropriate doses of 5 physiological solution for the constitution of said aliquot parts.

4. A kit in accordance with any one of claims 1 to 3, wherein said kit comprises said enzymes in lyophilised form subdivided into one or more aliquot parts, each 10 containing from 0.30 to 5,4 mg of Enzyme A, from 0,3 to 5,4 mg of Enzyme B, from 1,0 to 18 mg of Enzyme C and, optionally, three or, more aliquot parts of physiological solution of from 0.4 to 7.2 ml each.

5. Use of the enzymes reduced nicotinamide adenine 15 dinucleotide (NADH) reduced nicotinamide adenine dinucleotide phosphate (NADPH) (Enzyme B) and reduced gluathione (Enzyme C) for the preparation of a pharmaceutical composition in kit form for the treatment of retinitis pigmentosa by means of injection into the 20 retrobulbar tissue, said kit containing said enzymes in aliquot parts and interactive quantities appropriate for administering: a) Enzyme A at a concentration comprised between 0,25 and 0,35 mg in 0,4 ml of physiological 25 solution for three consecutive days, at monthly intervals, for three months and for each eye; b) Enzyme B, starting from the month following the last administration of Enzyme A, at a concentration of 0,25 to 0,35 mg in 0,4 ml of 30 physiological solution for three consecutive days, at monthly intervals, for three months and for each eye,; WO2005/120544 PCT/IT2004/000331 - 12 c) Enzyme C, starting from the month following the last administration of Enzyme B, at a concentration of 0,9 to 1,1 mg in 0,4 ml of physiological solution for three consecutive 5 days, at monthly intervals, for three months and for each eye.

6. Use of the enzymes in accordance with claim 5, wherein concentration of Enzyme A is 0,3 mg in 0,4 ml of physiological solution, the concentration of Enizyme B is 10 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.

7. Use of the enzymes in accordance with claims 5 or 6, wherein said kit comprises said enzymes in lyophilised 15 form, in quantities sufficient for at least one series of administrations of from a) to c) subdivided into aliquot parts containing - for each enzyme - a quantity of enzyme sufficient for one three-months, monthly, daily administration for each eye and, optionally, for the 20 constitution of said aliquot parts.

8. Use of the enzymes in accordance with any one of the preceding claims, wherein said kit comprises said enzymes in lyophilised form subdivided into one or more aliquot parts, each containing from 0,3 to 5,4 mg of Enzyme A, 25 from 0,3 to 5,4 mg. of Enzyme B, from 1,0 to 18 mg of Enzyme C and, optionally, three or more aliquot parts of physiological solution of from 0,4 to 7,2 ml each for each eye.

9. A method for the treatment of retinitis pigmentosa 30 that envisages the administration by means of injection into the retrobulbar tissue of: WO2005/120544 PCT/IT2004/000331 - 13 a) Reduced Nicotinamide adenine dinucleotide (NADH) (Enzyme A) at a concentration comprised between 0,25 and 0,35 mg in 0,4 ml of physiological solution for three consecutive days, at monthly intervals, for 5 three months and for each eye; b) Reduced Nicotinamide adenine dinucleotide phosphate (NADPH) (Enzyme B), starting from the month following the last administration of Enzyme A, at a concentration of 0,25 to 0,35 mg in 0,4 ml of 10 physiological solution for three consecutive days, at monthly intervals, for three months and for each eye; c) Reduced glutathione (Enzyme C), starting from the month following the last administration of Enzyme 15 B, at a concentration of 0,9 to 1,1 mg in 0,4 ml of physiological solution for three consecutive days, at monthly intervals, for three months and for each eye.

10. A method according to claim 9, wherein the 20 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. 25