WO1993001817A1

WO1993001817A1 - Antiviral compositions comprising fusidic acid, l-ascorbic acid and salicylic acid and derivatives

Info

Publication number: WO1993001817A1
Application number: PCT/GB1992/001386
Authority: WO
Inventors: Charles Lwanga Ssali
Original assignee: Charles Lwanga Ssali
Priority date: 1991-07-25
Filing date: 1992-07-27
Publication date: 1993-02-04
Also published as: AU2362692A; GB9116030D0

Abstract

A pharmaceutical composition for treating viral infections, notably the human immunodeficiency virus (HIV) comprises fusidic acid or a derivative or salt thereof such as sodium fusidate, L-ascorbic acid and salicylic acid or a pharmaceutically acceptable salt or derivative thereof such as acetyl salicylic acid.

Description

ANTIVIRAL COMPOSITIONS COMPRISING FUSIDIC ACID, L-ASCORBIC ACID AND SALIC ACID AND DERIVATIVES.

This invention relates to a synergistic pharmaceutical composition for use in the treatment and/or prevention of viral infections.

Numerous attempts have been made in recent years, to produce a pharmaceutical treatment to eradicate the human immune deficiency virus (HIV) from infected patients, and to produce a vaccine to prevent infection. The virus has however proved resistant to such attempts.

One of the difficulties which arises in trying to combat the HIV virus is that the virus invades and multiplies in host cells such as the white blood cells and the lymphocytes, and cannot easily be attacked within the host cell.

Fusidic acid (BP) SQ 16603, ent - 16α - acetoxy - 3β, llβ - dihydroxy -

4β, 8β, 14α - trimethyl -18- nor - 5β,

10a- cholesta (17z) - 17(20), 24 - dien - 21 - oic acid he ihydrate (C₃₁ H₄₈ Og. H₂0)

is an antimicrobial substance which can be produced by the growth of certain strains of Fusidium Coccineum (K.Tubaki).

The antibacterial activity of Fusidic acid, together with salts and solvates thereof, is described for example in GB-A-930786. It is active against Gram-positive bacteria and also against Gram-negative cocci. The most commonly used form is sodium fusidate, which has been found particularly useful against staphylococcal infections. It is sold under the trade mark FUCIDIN.

Compounds related to fusidic acid, notably the 3- and/or 11- dehydro derivatives and 24,25- dehydro derivatives and their salts, are disclosed in GB-A-992276. These derivatives have also been found to have useful antibacterial properties. Fusidic acid and its derivatives have not hitherto been thought to have any significant antiviral activity. It has however been found to attack HIV infected cells. This however does not solve the problem since if an infected cell is ruptured the virus is released into the blood stream and can attack other vulnerable cells.

I have however found that when Fusidic acid or a derivative thereof is administered together with ascorbic acid (vitamin C ) the two compounds together exhibit a synergistic action against certain viruses, notably the HIV virus.

My earlier UK patent specification GB-A-2224649 describes the treatment of HIV infected patients with a pharmaceutical composition comprising fusidic acid, or a pharmaceutically acceptable derivitive or salt thereof, and L-ascorbic acid (Vitamin C) .

The synergistic effect of the combination described in my earlier specification is thought to be due to the fact that a high level of ascorbic acid renders otherwise vulnerable cells such as leucocytes, and in particular T-Lymphocytes, resistant to a attack by fusidic acid if they are not already infected with the HIV virus, and also enhances their general immuno competence. Richman et al (Lancet 1988, i 1051-1052) demonstrated in vitro that fusidin has a toxicity against HIV infected cells, but no viricidal action per se. If therefore fusidic acid is administered in conjunction with high doses of ascorbic acid to an HIV infected patient, the infected cells can be selectively attacked and the immuno competent cells, rich in ascorbic acid, are able to resist attack by the released virus and to generate antibodies to combat the virus before it is able to attack another vulnerable host cell.

I have now found that a surprisingly improved synergistic effect against HIV infection can be obtained by administering fusidic acid or a salt or derivative thereof together with L-ascorbic acid and salicylic acid or a salt, ester or other derivative thereof such as acetyl salicylic acid (aspirin) .

Aspirin and other salicylic acid derivatives are widely used for their analgesic, anti-pyretic and anti inflammatory properties, but I have now found that they also have the effect of reducing the excretion of ascorbic acid in the urine and, at the same time, increasing its utilisa¬ tion in the body, probably by way of leucocytes. The exact mechanism by which this drug increases the speed of ascorbic acid uptake from body fluids are not fully known. It is however known that therapeutic doses of aspirin stimulate the hypothalamus to produce adrenocorticotropic hormone (ACTH) and increase the plasma levels of adrenocor- tic steroids. ACTH releases vitamin C from the adrenal cortex at the same time. Another likely mechanism is by displacement of steroids from plasma proteins, thus increasing their availability in circulation. The steroids have the dual effect of reducing the excretion of ascorbic acid in the urine and boosting its utilisation in body cells during stress. The similarity between the steroids and fusidic acid (which also has has a steroid structure),see for example GB-A-2224649) creates a favour¬ able synergism between the two in their action on the viral envelope.

There is thus produced a triple synergism between the three components of the composition of my invention which have been found to eradicate the HIV virus from infected patients. The postulated mechanism for the triple synergism is illustrated in more detail in the accompanying drawing.

Referring to the drawing, the box 10 schematically represents the infected cells of an immuno compromised aids victim. CD4T-lymphocytes 12 are attacked by the virus 14 and killed, releasing further viruses into the bloodstream. As viraemia increases, the blood becomes generally depleted in vitamin C, lymphocytes and antibodies.

The action of the three-component composition of the present invention is illustrated in box 20. Here a lymph gland is shown as 21, bone as 22, stem cells as 24, and CD4T-lymphocytes as 25.

Of the three components of the composition, the fusidic acid or derivative and the ascorbic acid act directly on the cells, while the salicylic acid or derivative acts indirectly, as will now be explained in more detail.

Salicylic acid and its derivatives stimulate the hypothalamus 16 to release ACTH through the pituitary 15. This stimulates the adrenal gland to increase blood steroids to drive ascorbic from the urine and gut and send it to tissues, leucocytes and stores to increase the body's general resistance to viral attack. Less ascorbic acid is excreted from the kidney 17 through the ureter 19, and the concentration in renal veins 13 is increased.

Fusidic acid or its derivatives or salts directly attack HIV infected cells, reducing their number. In conjunction with the blood steroids, it also attacks the virus directly, disrupting the viral envelope and, together with ascorbic acid, killing retroviruses.

In box 30 are shown immuno-competent cells, rich in ascorbic acid, which are able to produce antibodies to combat antigerms, while the ascorbic acid protects them from viral attack.

A pharmaceutical composition in accordance with the invention is preferably made up by forming a powder mixture of fusidic acid, suitably in the form of its sodium salt, ascorbic acid and aspirin. This may be dissolved in water or compressed into tablets with suitable adjuvants.

The ratio of L-ascorbic acid to sodium fusidate or other fusidic acid derivative in the composition is preferably from 1:1 to 3:1 by weight. Similarly, the preferred ratio of L-ascorbic acid to aspirin or other salicylic acid derivative is preferably 1:1 to 3:1 by weight. The preferred daily dosage ranges are 500 to 2000 mg of fusidic acid, 1000 to 4000 mg, preferably 2000 to 3000 mg, of ascorbic acid and 500 to 1500 mg, preferably 600 to 1200 mg, of aspirin, divided into four dosage units to be administered at six hourly intervals. In most cases the preferred unit dose for an adult of 75 to 85 kg body weight would be 250 mg fusidic acid, 500 mg ascorbic acid and 250 mg aspirin, dissolved in 50-80 ml water, this dosage being repeated every six hours to give a daily dosage of 4 grams of the composition (2g ascorbic acid and lg each of fusidic acid and aspirin).

In severe cases of the AIDS related complex, a composition having the above proportions is preferably administered at a daily dosage rate of about 75 mg/kg body weight, divided into four doses, for the first four weeks of treatment, this then being reduced to about 50 mg/kg body weight.

Other salicylate derivatives which may be used in place of acetyl salicylic acid include salts and esters of salicylic acid, in which substitution takes place in the carboxyl group, as well as esters in which the hydroxyl group of the salicylic acid is substituted. Compounds of particular interest include salicylic acid itself, methyl salicylate, sodium salicylate, sodium thiosalicylate, choline salicylate and magnesium salicylate.

Salts of fusidic acid other than the sodium salt can also be used in the composition of the invention. Apart from sodium, the diethanolamine salt is also suitable, as are the potassium, ammonium, triethyla ine, piperidine, morpholine, cyclohexylamine and monoethanolamine, all of which, unlike fusidic acid itself, are water soluble. Less water soluble salts which could also be considered are the calcium, magnesium, dibenzyl- ethylene-diamine, benzyl-β-phenyl-ethylamine and procaine salts.

Compounds closely related to fusidic acid which may also be used in accordance with the invention include 3-dehydrofusidic acid, 11-dehydrofusidic acid, 3,11- didehydrofusidic acid, 3-dehydro-24,25-dihydrofusidic acid, ll-dehydro-24,25-dihydrofusidic acid and 3,11-didehydro- 24,25-dihydrofusidic acid. These compounds can be obtained as for example as disclosed in GB-A-992276.

In a preferred treatment in accordance with the invention, recovery of the bone marrow cells may be assisted by administering folic acid (Vitamin B12).

The recommended initial term of treatment is 30 to 60 days at the end of which most of the viruses are likely to have been destroyed. The treatment is continued thereafter if necessary, for example for six, twelve or eighteen months.

As regards side effects, mild diarrhoea has been observed in some patients. Liver function tests should be carried out in cases of pre-existing liver dysfunction because of the action of fusidic acid.

Details will now be given of clinical trials carried out on HIV infected patients using the three- component composition of the invention.

Two treatment regimes are- illustrated, Treatment A being in accordance with my earlier application GB-A- 2,224,649, and exemplified here as a reference, and Treatment B being in accordance with the present invention. Treatment A:

The dosage ranges are as follows:

(1) Sod. Fusidate 500 mg ) Dose x 3 per day x 30 L-Ascorbic acid 1500 mg ) to 60 days or Dose x 4 per day x 30 to 60 days

(2) Sod. Fusidate 500 mg ) Dose x 3 per day. For L-Ascorbic acid 1000 mg ) 30 to 60 days or Dose x 4 per day. For 30 to 60 days

(3) Sod. Fusidate 250 mg ) Dose x 3 per day. For

L-Ascorbic acid 1000 mg ) 30 to 60 days or Dose x 4 per day. For 30 to 60 days

CHILDREN UNDER 10 YEARS

(4) Sod. Fusidate 125 mg ) Dose x 3 per day. For L-Ascorbic acid 500 mg 30 to 60 days or Dose x 4 per day. For 30 to 60 days.

Of the above, dosage (1) is preferred for adults of normal bodyweight, while (2) or (3) would be appropriate for patients of lower bodyweight and more particularly those more susceptible to side effects. In each case, the 4 per day dosage rate is intended for patients with AIDS symptoms while the 3 per day dosage rate is for patients who are HIV positive but do not yet have such symptoms.

A patient suffering from liver dysfunction, which might be aggravated by the fusidic acid, should generally be on dosage rate (3) . Treatment B:

A standard dose, to be administered at six hourly intervals, was prepared by mixing the following components: Powdered fusidic acid (sodium fusidate) 250 mg Powdered L-ascorbie acid (vitamin C) 500 mg Powdered acetyl salicylic acid (aspirin)250 mg The three powders were first mixed together and then dissolved in 50 to 80 ml of water. This standard dosage was for an adult of 75 to 85 kg bodyweight. The dosage could be reduced or increased depending on the bodyweight of the particular patient.

In severe cases of the aids related complex, a daily dose of 75 mg/kg bodyweight, divided into four doses, was used during the first four weeks of treatment, then reduced to 50 mg/kg bodyweight. No major side effects were observed, although some patients suffered from mild diarrhoea. The aspirin used was buffered, and produced no adverse side effects.

The 500 mg dose of sodium fusidate is therapeutically equivalent to approximately 714 mg of fusidic acid. The sodium salt, when administered orally, will be hydrolysed to the free acid in the stomach and digestive tract. Alternatively, buffered solutions of fusidic acid and ascorbic acid may be administered intraveneously or by means of suppositories. For oral administration, the medicament may be formulated in dosage units in capsules or in tablets or pills which may be enteric coated.

Where capsules are used, these may suitably contain separate pellets of the three components. These may have sustained release coatings to ensure that the three components are absorbed at comparable rates. Ascorbic acid in particular should preferably have a coating to prevent it being absorbed too quickly.

The composition of the invention may also be administered in syrup form, particularly to children. Example 1 (Reference) A 20-year old Ugandan female was taken ill;,while studying in the United Kingdom-, complaining first of weakness followed by recurrent fevers, body rash and abdominal pains. She lost weight rapidly a was admitted to hospital where test results showed that s was HIV-1 sero positive. Her leucocyte count was 2000 mm ³. She had a lymphopaenia and an erythocyte sedimentatio rate (ESR) of 57 mm/h (Westergreen) . She also suffered from oral and vaginal monilia and showed symptoms of deme tia. She was discharged from hospital and flown back to Uganda where Treatment A was started. After the start of treatment, her leucocyte count was measured and found to 1100 mm---'. Her condition remained poorly for about two weeks, because of pneumocystits carinii pneumonia and herpes simplex eruptions in the perineal region. A dosag rate of 50 mg/kg bodyweight, in divided doses in water, w maintained for eight weeks. At the end of this period th pneumonia had completely subsided and the temperature had settled down to normal. Her mental faculties greatly improved. The leucocyte count had risen to 2800 mm^-3, wit an increased number of lymphocytes present. She was also gaining weight and her appetite improved. At the end of the twelfth week of treatment, the leucocyte count was 350 πurT³ _wit 38% lymphocytes. The number of CD4T-lymphocytes was not known. At the end of the fourth month, the patien was well enough to walk four miles to her local clinic for a check-up. Her total leucocyte count was 4800 mm"³. Although she was still below her normal bodyweight, her mental state had returned to normal. Her only remaining complaint was a perineal sore from herpes simplex. At the end of 12 months from the start of treatment, the patient had made considerable progress and ceased to attend the clinic. She was last seen in apparently good health.

Example 2 A Ugandan male, aged 46, heterosexual, was married to the patient referred to in Example 6. When first seen, he complained of general weakness, frequent fevers, a body rash, diarrhoea and recurrent throat and pulmonary infections. He was found to be HIV-1 sero positive with a WBC count of 4600 mm^""3, a haemoglobin (Hb) level of 10 g/100 ml, ESR 20 mm/h, neutrophiles (N) 52%, lymphocytes (L) 42%, monocytes (M) 4% and oesinophiles (E 2%. He was put on Treatment A for eight months, and on Treatment B thereafter for a total treatment period of three years, and when last tested by the enzyme-linked immunosorbent assay (ELISA), he was found to be still slightly HIV positive. There were however no remaining symptoms or sign of HIV infection.

Example 3 An adult male, suffering from malabsorption syndrome, anemia, diabetes-mellitus and hypertension, was diagnosed HIV positive. After receivin Treatment A for eight weeks, his weight increased from 58 to 59 kg, ESR fell from 86 to 55 mm/h and the WBC fell from 11,000 to the normal 6,000 mm"³ The abnormally high count of white blood cells, mainly neutrophiles, was attributed to the aids related complex itself. Thereafter he was switched to Treatment B. The anemia improved, hypertension disappeared and blood pressure returned to normal. The patient's malabsorption improved remarkably and he stopped needing insulin for his diabetes. Thereafter he was sufficiently recovered to return to work.

Example 4 A male patient, suffering from fever and mycotic infection, had been diagnosed as having the HI aids related complex. After eight weeks of Treatment B, using the composition of the invention, his weight increas from 45 to 48 kg, ESR remained unchanged at 120 mm/h (Westergreen) , WBC increased from 6,000 to 6,150 mm^-3 and there was complete remission of fever and mycotic infection of the throat. This patient continued using the treatment for four months and then stopped, for personal reasons thought to be partly financial. Thereafter he remained in good health with remission of all HIV related symptoms.

Example 5 (Reference) A Ugandan female, aged 38, was diagnosed as HIV positive and complained of general weakness, loss of weight and dryness of skin. She also had an attack of herpes zoster which affected her arm. She was given Treatment A for two months but had to stop the treat¬ ment owing to financial difficulties which prevented her from purchasing further supplies. She died six months later.

Example 6 A Ugandan female, aged 40, is the wife of the patient referred to in Example 2. Her first complaint was general body weakness and polyuria. She was diagnosed as HIV-1 sero positive by ELISA and Western Blot Tests. She also had diabetes mellitus although there was no family history of diabetes. She started on Treatment B using the composition of the invention, at the same time as her husband and within five months her symptoms had disap¬ peared. She no longer required insulin, since there was no sugar in her urine and polyuria stopped. She continued the same treatment for a further 12 months and, when tested by the Kenya Medical Research Institute, was found to be HIV-1 negative by Western Blot. She was however to be slightly positive by ELISA but when tested some six months later she was found to be negative according to both tests.

Example 7 A 32-year old Ugandan female was the regular sexual partner of the patient described in Example 2. When her partner was diagnosed as HIV-1 positive, she was diagnosed as HIV sero negative but, since their sexual relations continued, her partner requested that she should begin the same treatment (Treatment B) . Subsequent sexual contacts were made using a condom and she was treated with the composition of the invention for 12 months. When last tested she had not sero-converted. She became pregnant after both partners had been treated in accordance with the invention for six months.

Example 8 A Ugandan male aged 28 contracted the aids related complex while living in the United Kingdom. He had initially been treated with AZT, which was later discontinued when his condition showed a toxic reaction with jaundice and anemia. When first seen by the present inventor, he was suffering from jaundice, fever, anemia and mental confusion. He had pneumocystitis carinii, which did not respond to antibiotics. He also had a persistent urinary infection. A lumbar puncture revealed meningitis. The patient had deteriorated to this serious condition while being treated in a London hospital. Medication against the opportunistic infections failed to eradicate them. He developed oral mycotic infection and a skin rash and had a rising temperature. It was at this stage that treatment with the composition of the present invention was suggested. At that time his WBC was very low, in the region of 2000 mm"³. After two weeks of Treatment B using the composition of the invention, he had no more pyrexia, his mental status improved and his urine culture was negative. The meningitis and pneumocystitis had been controlled. He continued to improve taking only the medi¬ cation of the invention and was thereafter monitored as an outpatient. The patient gained weight, through an improved appetite, and mental function improved. At the end of the fourth month of treatment, his total leucocyte count had risen to 3500 mm"³, there was no evidence of infection anywhere and his temperature remained normal.

Several of the patients described above took part in a more wide ranging controlled study involving 20 patients. All were Ugandan Africans of heterosexual background. There were four women and six men who received treatment. There were ten others used as controls who received placebo (glucose and calcium carbonate powder) . The administration of the drugs and placebo was randomised. Requirements for inclusion in the trial were as follows:

1. A positive test result by enzyme-linked immunosorbent assay or Western Blots.

2. A diagnosis of- AIDS, with at least one symptom.

3. Patients in coma or with signs of malignant disease were not taken on.

Participants were monitored for evidence of improvement, the disappearance of symptoms, fever, diarrhoea, skin rash, pneumocystitis carinii pneumonia, mental status improvement, a rise in a leucocyte count and a fall in erythrocyte sedimentation rate.

The end point of treatment was when symptoms and signs of Aids disappeared for at least six months. In some cases, treatment was carried on for 24 months despite the absence of signs and symptoms relating to Aids as in Case Nos. 4 and 9. All had normal white blood count and erythrocyte sedimentation rate.

Four of the patients received the minimum six months treatment and remained symptom free afterwards during the next 18 months of observation. Two patients, numbers 4 and 12, continued with the treatment for two years. They remained well and when tested again for HIV, number 12 was sero-negative but number 4 was weakly positive by Western Blot. None of the patients were tested for CD4 but the white blood count remained normal.

One patient who had improved after 12 weeks of treatment discharged herself. She was reported dead 16 months later having taken a sexual partner who had AIDS. She was excluded from the study. All the other patients observed strict abstention from unprotected sexual contacts.

Ten patients received the drug combination of Fusidin, Ascorbic Acid and Aspirin made up into a powder mixture of these, six patients (60%) are still alive at the time of writing these results. Their survival time ranges between 24-42 months. They are all symptom free. Two patients (20%) were lost to follow up between 12-22 months and their fate is unknown although they had shown improvement clinically at the time. Two patients (20%) died. One had a suspected brain tumour and died after four months of treatment. ^' The second one died after exposure to sub-zero temperatures in winter contracting pneumonia before his immunity had fully recovered.

Ten patients received Placebo - consisting of glucose and calcium carbonate powder mixture. Of these, nine patients (90%) died after periods varying between Ik to 8 months. One patient (10%) was lost to follow up after three months.

The results are summarised in more detail in the following table.

+ve (weakly)

table continued/..

Bottle Serial Treatment Patient's state From βtart of Therapy Sex Age Remarks No. for each of health SURVIVAL PERIOD IN Patient MONTHS

14 (Example 8) Died 9 months M 28 Died after exposure to sub zero winter weather before immunity recovery was complete

15 16 17

18 19 20

Claims

CLAIMS :

1. A pharmaceutical composition for treating viral infections, comprising fusidic acid or a derivative or salt thereof, L-ascorbic acid and salicylic acid or a pharmaceutically acceptable salt, ester or other derivative thereof.

2. A pharmaceutical composition according to claim 1 which comprises a fusidic acid salt selected from the sodium, calcium, magnesium, potassium, ammonium, diethanolamine, triethylamine, piperidine, morpholine, cyclohexylamine, monoethanolamine, dibenzylethylene- diamine, benzyl-β-phenylethylamine and procaine salts.

3. A pharmaceutical composition according to claim 1 which comprises a fusidic acid derivative selected from 3-dehydrofusidic acid, 11-dehydrofusidic acid, 3,11- didehydrofusidic acid, 3-dehydro-24,25-dihydrofusidic acid, ll-dehydro-24,25-dihydrofusidic acid, 3,ll-didehydro-24,25-dihydrofusidic acid and pharmaceutically acceptable salts thereof.

4. A pharmaceutical composition according to any preceding claim which comprises salicylic acid, acetyl salicylic acid, methyl salicylate, sodium salicylate, sodium thiosalicylate, choline salicylate and magnesium salicylate.

5. A pharmaceutical composition according to any preceding claim wherein the weight ratio of L-ascorbic acid to the fusidic acid, salt or derivative thereof is 1:1 to 3:1.

6. A pharmaceutical composition according to any preceding claim wherein the weight ratio of L-ascorbic acid to the salicylic acid, salt or ester thereof is 1:1 to 3:1.

7. A pharmaceutical composition according to any preceding claim comprising sodium fusidate and acetyl salicylic acid.

8. A pharmaceutical composition according to any preceding claim for use in combating the human immune deficiency virus (HIV) .

9. A method of treating viral infections which comprises administering a pharmaceutical composition as claimed in any preceding claim.

10. A method according to claim 9 wherein said composition is administered in daily doses of 500 to 2000 mg of fusidic acid, derivative or salt thereof, 1000 to 3000 mg of L-ascorbic acid and 500 to 1500 mg of salicylic acid, derivative or salt thereof.