WO1996006639A2

WO1996006639A2 - Novel drugs containing chelate-forming agents

Info

Publication number: WO1996006639A2
Application number: PCT/EP1995/003426
Authority: WO
Inventors: Serban Al. Bacanu; Iulian Ionescu; Sorin Sarzea; Stefan Teodor Tomas
Original assignee: Medico Pharma Vertriebs Gmbh; Sicomed S.A.
Priority date: 1994-09-01
Filing date: 1995-08-31
Publication date: 1996-03-07
Also published as: DE4431175A1; AU3519495A; WO1996006639A3

Abstract

The invention concerns novel drugs which contain chelate-forming agents and essential amino acids or their derivatives which are optionally complexed with bivalent metal ions. The novel drugs further contain bivalent elements or compounds of bivalent elements.

Description

New drugs containing chelating agents

The invention relates to new drugs, the chelating agents and essential amino acids or derivatives thereof, which are optionally complexed with divalent metal ions and contain divalent elements or compounds of divalent elements.

Chelating agents have long been used successfully as pharmaceuticals or components of pharmaceuticals in medicine or at least in the discussion, for example in the therapy of metal ion poisoning or in the inhibition of enzymes by binding cofactors. It is known that the steric conformation of the chelating agents and their ability to bind receptors have a strong influence on the therapeutic effects. However, many of the chelating agents known in the state of the art induce side effects which are contrary to a wide medical application (cf. for example Mazzarello et al., Schwe. Arch. Neurol. Psych. 131 (1982), 175-179). The furthest so far A common chelating agent used is azyclovir, which has already achieved considerable success in medical practice (eg Corey et al., New Engl. J. Med. 306 (1982), 1313). However, this medicinal product cannot be used without problems in all indications. For example, it is known that azyclovir in AIDS therapy can cause significant side effects. Another, known in the prior art drug is RODILEMID ^®, which has anti-viral, anti-inflammatoriεche, immunomodulatory and neurotrophic effects. In the past, this medicinal product has been particularly recommended due to its good tolerability and also due to its good therapeutic effect; see, for example, Dinu and Dinu, La Sante Publique (1985), 3 - 30. However, there is a need for further drugs which have the therapeutic properties of the above-mentioned chelating agents, moreover have better / different tolerance spectra and a further / different one Have application.

In many physiological processes, including in connection with the above-mentioned clinical pictures, play trace elements, e.g. Zinc, an important role. Changes in trace element concentrations (oligo element modulations) can be very useful in treatment, even if the exact mechanism of action of the oligo element modulation has not yet been fully clarified.

It was therefore an object of the present invention to provide medicaments with the positive properties described above, which can be used in a large number of indications or in other indications without causing harmful side effects and can act, inter alia, as chelating agents and / or oligo element modulators. This object is achieved by the embodiments described in the claims. The invention thus relates to a medicament containing

(a) a polyaminopolycarboxylic acid or a derivative or a complex thereof;

(b) an essential amino acid or a derivative thereof; and

(c) a divalent element or a compound of a divalent element; or

(a ') a polyaminopolycarboxylic acid or a derivative or a complex thereof; and (b ') a complex of an essential amino acid and divalent metal ions; with the exception of the combination

(a) EDTA-CaNa ₂

(b) Cysteine in a concentration of 0.38-2.3% by weight based on the sum of the three components

(c) calcium gluconate; and optionally a pharmaceutically acceptable carrier.

The pharmaceuticals according to the invention are characterized by a lack of toxicity when the usual concentrations are administered with optimal results. Moreover, it has been shown for an already known drug to a similar composition which is however not claimed as such in the present invention and named RODILEMID ^® that a prolonged administration carries no mutagenic potential. (Moralidhara et al., Food Chem. Toxicol. Dec. 12, 1991, 845-849). It is assumed that the mechanism of action of the pharmaceuticals according to the invention can be of different types. For example, the antiviral activity of the medicament according to the invention should be based on the fact that the withdrawal of zinc ions by the chelating agent brings about a disturbance in the zinc finger function of various retroviral proteins. The amino acid component in the embodiment as cysteine could play an important regulatory role between T cells and macrophages and thus develop immunomodulatory effects. In any case, there should also be synergistic effects of the individual components of the medicaments according to the invention, which predestine them for use in various therapies.

In a preferred embodiment of the medicament according to the invention, the polyaminopolycarboxylic acids mentioned above in (a) and (a ¹ ) are EDTA, DTPH, DCTA or derivative or complexes, preferably salts thereof, preferably EDTA CaNa2.

In a further preferred embodiment of the medicament according to the invention, the amino acid mentioned in (b) and (b ') is cysteine, histidine or methionine and, as a derivative thereof, a precursor of cysteine or of histidi or of methionine or N-acetylcysteine, N-acetylhistidi or N- Acetylmethionine.

In a further preferred embodiment of the medicament according to the invention, the divalent element mentioned in (c) is calcium and the derivative thereof is a calcium salt, preferably calcium lactate, calcium glucona or calcium levulinate.

In a further preferred embodiment, the i

(b ') called complex

(i) cysteine and copper;

(ii) serine and copper;

(iii) tyrosine and copper;

(iv) cysteine and zinc;

(v) serine and zinc; or

(vi) tyrosine and zinc.

In a further preferred embodiment, the medicament according to the invention contains at least one of the following mixtures 1. EDTA-CaNa2, N-acetylcysteine and calcium gluconate;

2. EDTA-CaNa ₂ , calcium gluconate and cysteine in a concentration greater than 2.3% by weight, based on the sum of the three components;

3. EDTA-CaNa2 and a complex of cysteine and copper;

4. EDTA-CaNa2 and a complex of serine and copper;

5. EDTA-CaNa2 and a complex of tyrosine and copper;

6. EDTA-CaNa2 and a complex of cysteine and zinc;

7. EDTA-CaNa2 and a complex of serine and zinc; or

8. EDTA-CaNa2 and a complex of tyrosine and zinc.

In a further preferred embodiment of the medicaments according to the invention, the polyaminopolycarboxylic acid mentioned in (a) and (a ¹ ) or the derivative or the

Complex thereof in a concentration of 90-95% by weight, the essential amino acid or the derivative thereof in a concentration of 0.3-3% by weight, the divalent element or the compound mentioned in (c) divalent element in a concentration of 3 - 7

% By weight, and the complex mentioned in (b ¹ ) in a concentration of

3.3 - 7% by weight.

The above percentages are based on the sum of the components (a), (b) and (c) or (a ¹ ) and (b ¹ ). The invention further relates to the use of the medicament according to the invention for the treatment of in particular viral, preferably retroviral diseases.

As already mentioned above, it is assumed that the antiviral mechanism of action is based on a disruption of the viral metal ion availability induced by the medicament according to the invention. With retroviruses in particular, it is known that the genomic RNA binds very strongly to nucleocapsid proteins, as a result of which the nucleocapsid structure is formed. The so-called NC protein is used required for RNA dimerization, also for encapsulation and for initiation of reverse transcription in human retroviruses. The NC7p7 protein of the HIV-1 nucleocapsid is necessary for the formation of infectious particles and contains two zinc fingers of the Cys-X2-Cys-X4-His-X4-Cys type. The medicament according to the invention presumably causes the zinc fingers to lose their biological abilities either through a steric change in the binding structure or through loss of the zinc ions, whereby in any case the three-dimensional structure of the nucleotide sequence may be changed, which ultimately results in the loss of the three-dimensional binding activity and with it the loss of infectiousness.

The invention further relates to the use of the medicament according to the invention and a medicament containing the composition for treating AIDS mentioned in the disclaimer mentioned above.

The invention further relates to the use of the medicament according to the invention for the treatment of inflammation, preferably polyradiculonevritis.

The invention further relates to the use of a medicament according to the invention as an immunomodulator; e.g. Immune stimulation in flu and paragrippal infections.

The invention also relates to the use of a medicament according to the invention for the treatment of autoimmune diseases.

In a particularly preferred embodiment, the invention relates to the use of the medicament according to the invention for the treatment of multiple sclerosis, Guillaume-Barre syndrome, as well as herpes simplex, herpes zoster and facial paresis. In a further preferred embodiment, the invention relates to the use of a medicament according to the invention for the treatment of neurodegenerative diseases.

Finally, the invention relates to the use of a medicament according to the invention and a medicament which has the composition mentioned in the above disclaimer as an anti-cancer agent.

Figure 1 shows the effectiveness of the pharmaceuticals according to the invention.

The examples illustrate the invention.

Example 1: Inhibition of HIV-1 Infections by EDTA CaNa2-Cvstein-Calciumσluconat (RODILEMID ^® )

The anti-HIV-1 activity and cytotoxicity of EDTA CaNa2 ~ cysteine calcium gluconate were measured in the MT-4 cell test (Harada et al., J. Immunol. Meth. 92 (1986), 177-181) and the results with the toxic and inactive anti-HIV-1 concentrations of four RODILEMID ^® derivatives (1A1H, 1A09, 1A2N, 1A1N), DMSO / H ₂ 0 (blind test, M) and azidothymidine (3 '-azido-2', 3 '-dideoxythymidine, AZT) compared.

The infection of the MT-4 cells was propagated to the IIIB strain of the HIV-1 virus (Popovic et al., Science 224 (1984), 497-500) in CEM cells. MT-4 and CEM cells (freely available, eg from ATCC) were grown in RPMI-1640 medium (Gibco, Karlsruhe, Germany) containing 20% by volume heat-inactivated fetal calf serum (FCS, Seromed, Berlin, Germany) and antibiotics had been added in the usual concentration. During the growth of the cells for the MT-4 cell test, these were grown in RPMI-1640 medium with 25 nM HEPES (2-4 (2-hydroxyethyl) -1-piperazinylethanesulphonate) and FCS. The MT-4 cell test was performed in 9-well microtiter plates. To measure the cytopathogenicity of the active ingredients, serial dilutions of RODILEMID ^® were incubated with 3 × 10 ⁴ MT-4 cells per well. Anti-HIV-1 activity was measured in the presence of the virus. The final virus concentrations were 10 TCID50, with one unit TCID50 being defined as the infectious dose in cell culture that reduced the number of living cells by 50%. Fresh media was added to each well three days after infection. Four μl after infection, 0.1 μCi H-thymidi (Amersham, Braunschweig, Germany) was added to each well. After 2 hours the cellular DNA was harvested on a glass fever filter and the incorporated HThymidine was determined in a scintillation counter using PPO, POPOP and toluene as a scintillator (Roth, Karlsruhe, Germany). The antiviral activity and the cytotoxic concentration of RODILEMID ^® were compared with the activities un Concentrations of the four RODILEMID ^® dse blank tests M compared to those of AZT.

For this purpose, the ³ H-thymidine incorporation in MT-4 cell DNA i the presence or absence of HIV-1 was plotted versus different concentrations of RODILEMID ^® and AZT (see Figure 1). The minimum toxic and maximum anti HIV-i concentrations of these compositions are shown in Table 1.

RODILEMID ^® was toxic to the MT-4 cells at a concentration of 1250 μg / ml (Figure la, dashed line). A RODILEMID ^® concentration of 625 μg / ml had no influence on the cell growth and at this concentration a strong anti-HIV-1 activity could be determined via a strong ³ H thymidine incorporation (FIG. 1 a, solid line). An anti-HIV-1 activity of RODILEMID ^® could be determined at concentrations between 625 μg / ml and 39 μg / ml. AZT was toxic to those in MT-4 cells at a concentration of 250 µM (Figure 1b, dashed line). The anti-HIV1 activity of AZT could be measured in the range of 25000 nM and 25 nM (Fibur lb, solid line). A measurable difference in the anti-HIV-1 activity of RODILEMID ^® and the derivatives could not be determined. In terms of toxicity, only 1A1H is less toxic to MT-4 cells than RODILEMID ^® : MT-4 cells died at a concentration of 2500 μg / ml 1A1H. The three other derivatives 1A09, 1A2N and 1A1N were toxic in the same range as RODILEMID ^® (1250 μg / ml).

The selectivity index SI (see Table 1) defined as 100% cell toxicity concentration divided by 100% antiviral concentrations for a compound / composition was highest for AZT (10000). Thus, with an SI of 32, RODILEMID ^{® was} about 300 times less active than AZT and twice as active than the RODILEMID ^® derivative 1A1H.

Table 1

Toxicity and anti-HIV activities of RODILEMID® derivatives and non-RODILEMID® derivatives

Substance toxicity antiviral SI ¹ effectiveness

Rodilemid 1250 μg / ml 39 μg / ml 32

Rodilemide derivatives:

1A1H 2500 ug / ml 39 ug / ml 64

1A09 1250 μg / ml 39 μg / ml 32

1A2N 1250 μg / ml 39 μg / ml 32

1A1N 1250 μg / ml 39 μg / ml 32

other compositions: M> 25 μl _2

AZT 250000 nM 25 nM 10000

¹ SI: Selectivity index is defined as 100% cell toxicity concentration divided by 100% antiviral concentrations for a compound / composition No detectable antiviral activity and therefore no SI value.

Example 2: Treatment scheme for the use of the inventive drug in MS, AIDS. Herpes Simplex, Herpes Zoster and the Guillaume-Barre-Svndrom

The treatment process consists of long-term treatment with the drug and depends on the development of the patient's clinical symptoms and other common para-clinical parameters.

The treatment regimen includes active periods that alternate with periods when the drug is not being used. Treatment is characterized by a phase of long alternating periods and a phase of short ones

SPARE BLÄΓΓ (REÖEL 26) alternating periods marked. The daily doses vary between 7 and 70 mg per kg body weight, which are administered as intramuscular, intravenous injections or as an oral dose. If oral therapy is used, the dose described above can be doubled. After a break of 20 to 25 days, the therapy is started again cyclically. After therapy cycles of 12 to 15 days, a longer break of, for example, three months can be inserted, the length of which in turn depends on the clinical status and the usual para-clinical parameters.

The therapy begins with the appearance of the disease symptoms to be treated, in the case of multiple sclerosis of a relapse, 15 to 70 mg of the medicament according to the invention being administered per kg body weight as intramuscular or intravenous injections for 15 to 30 days. Thereafter, the treatment with doses of 7 to 35 mg of medicinal product per kilogram of body weight is continued for 3 to 6 months, it being possible to use the administration routes described above. Depending on the patient's clinical picture and the usual paraclinic parameters, the treatment can be repeated after one to six months.

During treatment, a strict diet must be followed, the task of which is to increase the effectiveness of the drug. The mechanism of action discussed above and pharmacological studies indicate that the exogenous supply of divalent metal ions greatly reduces the biological effectiveness of the drug. Therefore, the consumption of the following foods during the active treatment phase must be avoided in any case: grapefruit, lemons, oranges, cabbage, salads, leafy greens, milk derivatives, cheese, yogurt, mineral water, grapefruit juice, lemon juice, orange juice, beans, peas, soy, mushrooms, Tomatoes, carrots, beetroot, radishes, celery, cucumber, Onions, wheat derivatives, milk pastries, liver, kidney.

In contrast, apples, pears, bananas, cherries, peaches, watermelons, grapes, coffee, tea, cereals, potatoes, white beets, eggs, veal, poultry, fish, fat, rye bread, wheat bread, wheat starch, wheat, oatmeal, paddy rice, husked rice, rice starch, potato starch, flour are consumed.

Example 3: RODILEMID ^® as an AIDS therapeutic

First, the toxic activity of RODILEMID was determined: in rats the D50 is 4790 mg / k body weight, in mice even 5940 mg / kg body weight.

Chronic toxicity from intramuscular injections in rats and beagle dogs showed that up to doses of 350 mg / kg body weight there was no toxicity, no mutagenic and no embryotoxic effects. This dose is 20 times higher than the therapeutically effective dose.

These results confirm the good uses of RODILEMID ^® for use as a medicine in humans, for example in AIDS therapy.

Clinical trial

In the first stage of therapy, eight AIDS patients were checked clinically and immunologically. The Abbot kit was used for this. All samples were tested again using either the indirect or competitive Wellcozyme or the Abbot kit and subsequently by Western blot. The patients consistently showed a T cell count of around 450 per mm at the start of the studies. The patients were only called to the clinic for taking samples. The immunological monitoring was carried out by the Total lymphocyte count and T4 and Tg subpopulations monitored using the rosette method.

RODILEMID ^® therapy started on the first day of immunological tests by intramuscular injection per day over a period of 10 days. After one month, the patients received an oral dose a day, 14 days a month. Therapy had to be stopped four months later. It was then continued with 10 AIDS patients, seven of whom belonged to the first group examined and three new patients were added. These patients were given 10 doses per month, with only one dose being given each day. The immunological examinations were carried out once every 45 days after the restart of the treatment and the clinical monitoring has taken 30 or 18 months (for the three newly added patients) since the start of the study.

In parallel with these studies, a group of seven HIV-1 infected patients, identified as such 1 to 4.5 years earlier, were observed immunologically and clinically after starting oral therapy with RODILEMID®. The therapy included the administration of 10 ampoules per month, one ampoule being administered per day.

10 AIDS patients were observed as a control group. Their immunological development was examined simultaneously with the group treated with RODILEMID ^® . In addition, four children between the ages of 1 and 1.5 years who had been transmitted HIV-1 horizontally were observed for 18 months with RODILEMID ^® . The children were given one ampoule a day 10 days a month. During the active RODILEMID ^® therapy lasting 10 days per month any calcium intake, including dairy products, has been prevented.

A significant increase in total lymphocyte counts and an increase in all T lymphocyte subpopulations examined were observed in the group of eight AIDS patients after four-month treatment with RODILEMID ^® , which started with a series of 10 intramuscular injections and continued with oral administration . The T ₄ lymphocyte population rose to an average value of 1000 per mm 2. At the same time, an improved clinical appearance of the patients was observed.

As in the case of the already known indications for treatment with RODILEMID ^® , HIV, herpes zoster and viral polyneuritis, the development of the Tg subpopulations takes the expected course and grows in the course of the treatment. Furthermore, an increase in the T-lymphocyte subpopulations and a significant decrease in the IgG antibodies from an average of 3200 mg% to 1680 mg% after 5 months of RODILEMID, treatment were observed. During the second phase of RODILEMID® therapy, which was resumed after 1 month, an increase in the total number of lymphocytes in all subpopulations was observed in all 10 patients to whom RODILEMID ^{® was} administered orally, ie the seven patients from the first group and the three patients who were added later. However, this increase was less significant than that during the first phase, when the drug was administered intramuscularly. This is probably due to the slightly lower dose. After 3 months of oral RODILEMID ^® treatment, the average value for the T4 subpopulations increased from 408 / mm to 640 / mm.

After the forced termination after 10 months after the first therapy phase, one patient died 4 months after the treatment was stopped. She had been released from the hospital after significant improvements in the clinical picture were observed.

After resuming treatment with the 10 AIDS patients, one of the patients stopped the treatment after taking four series of 10 ampoules and died 10 months later. At the time the treatment was discontinued, it had a good clinical and immunological appearance. The remaining nine patients currently have a good clinical presentation. From a clinical point of view, the nine patients were in the seventh stage of the Walter Reed (WR) scale. After 30 months of clinical monitoring for the seven patients and 18 months for the three additional patients, these show a progress from the original assessment as the WR 4 stage to the WR 2 stage after the end of the treatment.

As general clinical observations it should be noted: The adenopathies are fluctuating. The flare-up of herpes infections was observed very rarely and only occurred with reduced strength. Herpes zoster infections were cured within six to seven days, and patients responded well to both specific and complementary treatments for opportunistic infections, gaining weight.

In contrast to the data presented above, the AIDS patients from the control group showed the following symptoms: progressive reduction in the total number of lymphocytes and in all T-lymphocyte subpopulations. The number of T ~ cells in the most advanced clinical picture decreased by about half after 10 months. Five patients in this group have since died and two patients who have been treated with AZT also showed a progressive decrease in T4 ~ lymphocytes. Thereby values of 130 / mm ³ and 250 / mm ³ reached, one of the two patients has since died. A somewhat slowed decrease in T4 lymphocyte counts on the hold was observed in this patient. Two of the patients are in the WR-6 phase.

None of the seven HIV-1 infected patients who were treated orally with RODILEMID ^® (30 to 18 months) have meanwhile reached a clinical clinical picture. On the WR scale, they are on average between WR 1.66 and WR 1.33. The immunological examination showed that increases in the total number were observed in all T-lymphocyte subpopulations, with mean values of the T4-lymphocyte numbers from 526 / mm to 825 / mm being observed after four months of treatment.

AIDS kills children relatively soon. Of the four

Children with the AIDS appearance who take orally

RODILEMID ^® has been treated for 26 months, one has died, but the others are still alive.

In summary, it can be stated here that RODILEMID ^{® has} unambiguously contributed to an improved clinical appearance and an extension of the lifespan of AIDS patients. In HIV-1 infected patients the transition to the disease stage could be prevented or at least slowed down. Treatment is easy to perform, inexpensive, and no toxic or other side effects have been observed. The above-described differences in oral or intramuscular use in connection with the non-toxicity of RODILEMID ^{® leads to} the conclusion that RODILEMID ^® can be administered in double dose in adults, which could lead to even better treatment results.

Claims

Patent claims

1. Medicament containing

(a) a polyaminopolycarboxylic acid or a derivative or a complex thereof;

(b) an essential amino acid or a derivative thereof; and

(c) a divalent element or a compound of a divalent element; or

(a ') a polyaminopolycarboxylic acid or a derivative or a complex thereof; and (b ¹ ) a complex of an essential amino acid and a divalent metal ion; with the exception of the combination

(a) EDTA CaNa ₂ ;

(b) cysteine in a concentration of 0.38-2.3% by weight based on the sum of the three components;

(c) calcium gluconate; and optionally a pharmaceutically acceptable carrier.

2. Medicament according to claim 1, wherein the polyaminopolycarboxylic acid mentioned in (a) and (a ') is EDTA, DTPA, DCTA or a derivative or a complex or a salt thereof, preferably EDTA-CaNa2.

3. Medicament according to claim 1 or 2, wherein the amino acid mentioned in (b) and (b ') is cysteine, histidine or methionine and the derivative thereof is a precursor of cysteine or histidine or of methionine or N-acetylcysteine, N-acetylhistidine or Is N-acetylmethionine. Medicament according to one of claims 1 to 3, wherein the divalent element calcium mentioned in (c) and the derivative thereof is a calcium salt, preferably calcium lactate, calcium gluconate or calcium levulinate.

Medicament according to one of claims 1 to 3, wherein the complex mentioned in (b ¹ ) from

(i) cysteine and copper;

(ii) serine and copper;

(iii) tyrosine and copper;

(iv) cysteine and zinc;

(v) serine and zinc; or

(vi) tyrosine and zinc.

Medicament containing at least one of the following mixtures

1. EDTA-CaNa ₂ , N-acetylcysteine and calcium gluconate;

2. EDTA-CaNa2, calcium gluconate and cysteine in a concentration greater than 2.3% by weight, based on the sum of the three components;

3. EDTA-CaNa2 and a complex of cysteine and copper;

4. EDTA-CaNa2 and a complex of serine and copper;

5. EDTA-CaNa2 and a complex of tyrosine and copper;

6. EDTA-CaNa2 and a complex of cysteine and zinc;

7. EDTA-CaNa2 and a complex of serine and zinc; or

8. EDTA-CaNa2 and a complex of tyrosine and zinc.

Medicament according to one of claims 1 to 6, wherein the polyaminopolycarboxylic acid or the derivative or the complex thereof mentioned in (a) and (a ¹ ) in a concentration of 90-95% by weight, the essential amino acid mentioned in (b) or the Derivative thereof in a concentration of 0.3-3% by weight, the divalent element mentioned in (c) or the compound of a divalent element in a concentration of 3-7% by weight, and the complex mentioned in (b ¹ ) occurs in a concentration of 3.3-7% by weight.

8. Use of a medicament according to one of claims 1 to 7 for the treatment of viral, preferably retro-viral diseases.

9. Use of a medicament according to one of claims 1 to 7, and a medicament containing the composition mentioned in the disclaimer of claim 1, for the treatment of AIDS.

10. Use of a medicament according to one of claims 1 to 7, preferably of herpes simplex, herpes zoster and facial paresis.

11. Use of a medicament according to one of claims 1 to 7 as an immunomodulator.

12. Use of a medicament according to one of claims 1 to 7, and a medicament containing the composition mentioned in the disclaimer of claim 1 for the treatment of autoimmune diseases.

13. Use according to claim 12, wherein the autoimmune disease is multiple sclerosis or Guillaume-Barre syndrome.

14. Use of a medicament according to one of claims 1 to 7 for the treatment of neurodegenerative diseases.

15. Use of a medicament according to one of claims 1 to 7 and a medicament containing the composition mentioned in the disclaimer of claim 1 as an anti-cancer agent.