Dear Sevda Demir,

herein I am sending You the answer as follows: You should use Acyclovir-Sodium, with much higher water solubility, up to 50 mg/ml. So you will get Protocol for preparation (solubilization) of Acyclovir - Sodium. Further is the protocol for calculation of Cytotoxicity (cc50) of Acyclovir-Sodium on Vero cells, as well as

Antiviral (standard) assay. Finsihed is with Selectivity index (SI) showing the real value of Acyclovir - Sodium. I sincirely hope, that You will be sucessfull. Please inform me about Your work, together with some new problems. Sincerely Yours,

Dr. Bratko Filipic

Email: [email protected]

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Preparation of Acyclovir - Sodium (AS)

1. Put 50 mg of AS crystals into 25 ml graduated Falcon tube.

2. With pipet with 1000 μl (1 ml) tip add 1 ml of DD - or MQ - water several times push through and put Falcon tube into Water-Bath (37oC) for 30 minutes.

3. Every 5 minutes push through the suspension.

4. After reconstitution the AS solution has pH 10.5 - 11.7

5. To 1.0 ml of AS add 4.0 of DMEM and mix well.

6. With the addition of 1M HCl make final pH 7.2

7. Add final 5.0 ml of DMEM measure pH and sterylise by 0.2 Syringe filter

8. Put per 1.0 ml into sterile Tube and store at -20 oC till use

Cytotoxicity Assay

As for the Acyclovir - Sodium (AS) cytotoxicity evaluation:

1. Vero cells (2.5×105 cells/mL) were seeded into 96-well plates per 100μl of DMEM + cells and incubated overnight at 37°C.

2. Upon 80% confluence, the cells were treated as follows:

2.1.Into 1st , 2nd, 3rd and 4th well (A,B,C,D) in row 100 μl from 10 ml DMEM containg 0.5 mg/mL of AS.

2.2. Serrialy transfer 100 μl from 1st to 11th well.

2.3. From 11th well 100 μl discharrd.

2.4. 12th column 12/ wells: A, B, C, D, E, F, G, H with cells with only growth medium (DMEM) were used as negative control.

2.5. After incubation of about 72h, the growth medium was discarded and replaced with 30 μL of 3(4,5-dimethythiazol-2-yl)- 2,5-diphenyl tetrazolium bromide or MTT solution and incubated for 3h.

2.6. After that, the MTT solution was discharged and formazan crystal was dissolved using 100 μL of dimethyl sulphoxide (DMSO) to lyse the cells.

2.7. Colour development was detected using a microplate reader (BioRad Model 680, USA) at 540 nm.

2.8. The cytotoxic concentration, CC50, was defined as the concentration of AS that reduced the population of the treated cells to 50% by testing them thrice with 96 well plates and using 4 replicates for each well.

Antiviral Assays

The HSV strain KOS-1 was isolated from the infected Vero cells using standard protocols [1]. Screening for antiviral activity was performed using three different treatments .

1) Post-treatment (C+V)+E: The Vero cells (C) were infected with virus (V) and underwent 2h of incubation, then the mixture of SA (E) and DMEM was inoculated on the plate per 100 μl and incubated for 48h.

2) Pre-treatment (C+E)+V: The cells (C) were incubated with SA (E) for 24h before they were inoculated with virus (V).

3) Virusidal C+(E+V): Virus (V) with SA (E) was incubated for several hours (0, 1, 2, and 4h) before it was inoculated on the cells per 100 μl (C).

The SA concentration tested was twice lower than the CC50 value in order to reduce the possibility of toxicity towards the cells.

The numbers of plaques were counted and the antiviral activity was determined using the formula below:

The percentage = Average of control plaque - Average of tested plaque

of antiviral ----------------------------------------------------------------- × 100%

activity Average of control plaque

with Average of control plaque = Non-treated infected cells, Average of tested plaque = SA treated infected cell. The effectiveness of SA as an antiviral agent expressed as selectivity index (SI) calculated as below:

Selectivity Index (SI) = Cytotoxicity concentration (CC50)

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Effective concentration (EC50)

Dear Filipic,

Thank you for your answer.

Dear Sevda Demir,

herein I am adding the Protokols (Examples), how to prepare Acyclovir Sodium from Acyclovir. But important: THESE WORKS CAN PERFORM THE QUALIFIED PERSON ONLY. THEY CAN BE DANGEROUS IF WORKS PERFORM UNQUALIFIED PERSON!!

Sincerely, Yours,

Dr. Bratko Filipic

Email: [email protected]

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The following examples further illustrate the invention.

EXAMPLE 1

Acyclovir (90 g; 0.376 moles) is suspended in distilled water (480 ml) in a 1 liter glass round-bottomed flask. A solution of sodium hydroxide (15.9 g; 0.397 moles) in distilled water (100 ml) is dropped therein at a temperature of 20° C. Decolourizing charcoal (0.8 g) is added, and the solution is filtered, washing the filter with distilled water. The resulting solution is dropped slowly into a 5 liter glass round-bottomed flask containing acetone previously cooled at a temperature of 10° C. (2800 ml). The mixture is kept under stirring for some hours, after which the resulting crystalline white product is filtered with suction. The filtration cake is washed with cold acetone.

The product is recovered and dried under vacuum at a temperature of 40-45° C. for 16 hours.

83 g of product having 12.45% K.F. are obtained.

EXAMPLE 2

A sodium hydroxide 29% w/w solution (29.8 g; 0.209 moles) is dropped into a 500 ml glass round-bottomed flask containing a suspension of Acyclovir (50 g; 0.209 moles) in distilled water (75 ml). The resulting solution is filtered through fluted filter paper and dropped into a 2 l glass round-bottomed flask containing isopropanol previously cooled at 0-5° C. (850 ml). After some hours under stirring, the resulting crystalline product is filtered and the filtration cake is washed with cold isopropanol, then dried under vacuum at 50° C. for 16 hours.

58.2 g of a crystalline white product having 13% K.F. are obtained.

The Examples 3 and 4 show the interconversion between the forms freeze-dried Acyclovir sodium salt and Acyclovir crystalline dihydrate sodium salt.

EXAMPLE 3

Sodium hydroxide (3.36 g; 0.084 moles) is dissolved in distilled water (50 ml) in a 250 ml glass round-bottomed flask. Acyclovir 2/3 H2 O (20 g; 0.084 moles) is added and the resulting solution is concentrated to dryness to obtain a residue, which is taken up with methanol (100 ml) and stirred at room temperature for some hours. The resulting crystalline solid is filtered and washed with some methanol.

The residue is recovered and dried under vacuum at a temperature of 55° C. for 16 hours.

16.5 g of a crystalline white product having 1.8% are K.F. obtained.

A sample of the same product, after exposure to air for 8 hours, shows a 13.5% water content.

EXAMPLE 4

Acyclovir (200 g; 0.844 moles) is suspended in distilled water (300 ml) in a 1 liter glass flask and a sodium hydroxide 30% w/w solution (83.5 ml) is dropped therein to obtain complete dissolution at pH 12.5, measured at a temperature of 22° C. The solution is dropped into cold isopropanol (3200 ml) and the resulting solid crystalline product is recovered and dried. 217 g of crystalline sodium salt having 13.6% K.F. are obtained.

20 g of said product are redissolved in distilled water (150 ml) and distributed into vials, each containing 12 ml of the obtained solution. Said vials are freeze-dried.

13 Vials of freeze-dried acyclovir sodium salt, each of about 1.3 g, are thereby obtained.

The water content of one of said vials, determined immediately after opening, is 2.6%. The same sample of freeze-dried product, after exposure to air for 60 min. shows a 14.8% water content (stable even after 8 hours).

EXAMPLE 5 Preparation of Crystalline Apyrogenic Acyclovir Dihydrate Sodium Salt

Dry acyclovir (K.F. 5%) (40 kg) and demineralized water (60 l) are placed into a 250 l stainless steel reactor. A sodium hydroxide 30% aqueous solution (25.3 kg) is dropped therein with stirring until complete dissolution, at a temperature of 20° C.

Said solution is filtered through a millipore 0.22 μm filter in an enameled reactor (800 l) containing isopropanol (700 l), keeping stirring for some hours, then centrifuging the resulting crystalline white product. The product is then washed with isopropanol, recovered and dried at a temperature of 60° C. for 16 hours under vacuum.

About 44 kg of crystalline apyrogenic acyclovir dihydrate sodium salt (L.A.L. Test