A Novel Bimodal Release Strategy for Atazanavir And Ritonavir: Mini-Tablet in Capsule Formulation with Ph-Dependent Coatings and Simultaneous Estimation Via UV Spectroscopy

The Human Immunodeficiency Virus persists as one of the major global health issues, with a large number of people being infected worldwide. Infection with HIV is characterized by progressive deterioration in immune function, particularly the loss of CD4+ T-cells, which reduces resistance to opportunistic infections [1]. This is supported by the fact that effective management of HIV requires a suppressed viral load; interruption of ART is associated with resurgence of viral load, reducing treatment efficacy and increasing drug resistance [2]. One of the important factors in HIV therapy is the diurnal variation in immune cell activity. It was shown that the activity of CD4+ T-cells is low during early morning hours and increases gradually throughout the day [3]. This temporal variation in immune function poses a challenge, as it demands consistent antiretroviral drug availability to support the immune system when its activities are low [4]. For the solution of such problems, drug delivery in the bimodal form of immediate and controlled release has been developed which gives an initial surge to cater for immune deficiency at the beginning of the day with continued release to maintain therapeutic levels throughout increased activity of CD4+ T cells. HAART, or highly active antiretroviral therapy, combines antiretroviral drugs that attack the virus at different stages in its lifecycle, and is extremely effective. These include Atazanavir and Ritonavir, which are considered to be complementary in their pharmacokinetic profiles. Atazanavir is a protease inhibitor that requires the boosting action of Ritonavir to reach optimal therapeutic concentration. However, long-term use of such drugs, like many other antiretrovirals, is associated with considerable adverse side effects, including hepatotoxicity, dyslipidemia, gastrointestinal disturbances, and cardiovascular risks [5]. In order to minimize these side effects and enhance patient compliance, we propose a strategy of reducing the overall doses of Atazanavir and Ritonavir without compromising their therapeutic efficacy [6, 7]. This is achieved by using a loading dose and maintenance dose, which are incorporated as mini-tablet-in-capsule bimodal DDS. The loading dose ensures an immediate therapeutic effect at times of immune vulnerability, while the maintenance dose gives steady-state levels to support CD4+ T-cell activity throughout the day. It is further envisioned that such chronobiologically timed drug release according to the body's immune rhythms will lead to better therapeutic outcome with reduced toxicity and improved long-term adherence to therapy. This work aims at the development and in-vitro evaluation of this new bimodal mini-tablet-in-capsule drug delivery system combining Atazanavir and Ritonavir for an effective viral load suppression and the mitigation of temporal dynamics of immune function besides reducing the side effects of HIV therapy.

MATERIALS AND METHODS:

MATERIALS:

Atazanavir (ATV), Ritonavir (RTV) was gifted from Sai Mirra Innopharm, Chennai, India. β Cyclodextrin was procured from Manus Aktteva Bio Pharma LLP and Poloxamer 188, Starch 1500 (pregelatinized starch) were gifted by Corel Pharma Chem. Hydroxypropyl Methylcellulose K4M, K15M and K100M CR Premium were gifted by Colorconasia Pvt. Ltd. Eudragit L 55-100, S 100 were procured from Evonik Rohm GmbH, Pharma polymers, Germany. Coloring agents Quinoline Yellow WS and Sunset Yellow Supra were procured from Color Trendz. Solvents like dichloromethane and isopropyl alcohol along with purified water was supplied by Lab Chemicals while sodium citrate was procured from Corel Pharma Chem.

Dose calculation:

The dose for the combination therapy of Atazanavir and Ritonavir was calculated in order to minimize the side effects, reduce production costs, and enhance the adherence of the patients. A proposed reduction in the currently marketed dosage of 300 mg/100 mg was developed by utilizing a loading dose meant for the rapid achievement of therapeutic plasma concentrations, followed by a maintenance dose that will maintain time-dependent drug levels. These reduced doses achieve similar therapeutic results as the usual doses through pharmacokinetic adjustments, thus improving the patient's compliance.mThe dosing schedule can be calculated based on two important formulae: one for the loading dose and one for the maintenance dose.

              Loading dose = Cp x Vd/F

Where,

Cp is the target plasma concentration,

Vd is the volume of distribution,

F is the bioavailability of the drug.

To determine the loading dose of Atazanavir, Cp (target plasma concentration) [8] = 150 ng/ml = 0.00015 g/L = 1.5×10−4g/L.

Vd (volume of distribution) [9] = 1.604 L/kg. For an average human weight of 75 kg, the total volume of distribution is 1.604 x 75=120.3L.

F (bioavailability) [10] = 60% to 68%.

By using the formula,

          L.D = Cp x Vd/F

= (1.5 x ?10?^ (-4) g/L x 120.3L)/0.6

=0.018045/0.6

=0.030075g

 = 30.08mg.

Hence, to account for potential variability in patient pharmacokinetics, the calculated loading dose of 30.08 mg has been approximated to 35 mg, while remaining within a safe and effective dosing range.

To determine the loading dose of Ritonavir,

Cp (target plasma concentration) = 0.06 mcg/ml = 0.00006 g/L = 6×10−5g/L.

Vd (volume of distribution) [11] = Ritonavir's volume of distribution is estimated at 0.6 L/kg. Assuming an average human body weight of 75 kg,

Vd = 0.6 L/kg x 75kg = 45L.

F (bioavailability) [12] = The bioavailability of Ritonavir is typically around 60% or 0.60.

By using the loading dose formula,

L.D = Cp x Vd/F

= (6 x ?10?^ (-5) g/L x 45L)/0.6

= (2.7 x ?10?^ (-3) g)/0.6

= 4.5 x ?10?^ (-3) g = 4.5mg.

The loading dose of Ritonavir is approximated to 5mg. This slight adjustment simplifies the dosing while maintaining therapeutic relevance, ensuring that the plasma concentration reaches the desired level efficiently.

Since initial loading dose concentration is calculated. Now, to maintain the therapeutic concentration, the maintenance dose (MD) is calculated using the formula,

           Maintenance dose = Cp x Cl x τ/F

Where,

Cp is the target plasma concentration,

Cl is the clearance rate of the drug,

τ (tau) represents the dosing interval,

F is the bioavailability of the drug.

To determine the maintenance dose of Atazanavir,

First will determine the given parameters,

Cl (Clearance) = Vd x Ke = 120.3L x 0.08662 hr−1

Cl = 10.42L/hr.

τ = 24 hrs (24 hours for once-daily dosing)

F = 68%.

By using the formula,

M.D = Cp x Cl x τ/F

=1.5 x 10-4g/l) x (10.42 L/hr)x 24hr0.68

= 3.75072 x 10-2g0.68

= 0.0551g = 55mg.

Here, hence the calculated maintenance dose of Atazanavir is approximately 55 mg. This dose ensures that the target plasma concentration is maintained over the 24-hour dosing interval, taking into account the drug’s clearance and bioavailability.

To determine the maintenance dose of Ritonavir,

Given parameters,

Cl (Clearance) = 9L/hr.

τ = 24 hrs (24 hours for once-daily dosing)

F = 60% to 68%, F = 0.60.

By using the formula,

M.D = Cp x Cl x τ/F

= 6 x10-5glx9Lhrx 24 hr0.6

= 0.01296g0.6

= 0.0216g = 21.6mg.

The calculated maintenance dose of Ritonavir is approximated to 25mg. With the loading dose and maintenance dose of Atazanavir at 35mg and 55mg and Ritonavir at 5mg and 25mg respectively is fixed, the next phase involves formulating IR and CR mini-tablet.

Preparation of immediate release mini-tablet (IRMT):

The ingredients were weighed as per the formulation requirement. For L1 and L3, mixture of Atazanavir and Ritonavir with β-cyclodextrin (10% and 20%) were taken under solvent evaporation method in order to improve their solubility. β-cyclodextrin was dissolved in the methanol and drugs were added to this solution, and the solvent was removed under rotary vacuum. For L2 and L4, Atazanavir and Ritonavir were combined with Poloxamer 188 (10% and 20%) in a similar manner, later, the solvent was evaporated. The complex mixture were mixed with Starch 1500 (as binder), Crospovidone XL 10 (as disintegrant), Aerosil (as glidant), and Magnesium Stearate (as lubricant). The mixture was thoroughly homogenized and compressed into 100 mg mini-tablets utilizing a 6.5 mm punch. The weight and thickness of the tablets were controlled during compression to ensure uniformity.

Preparation of controlled release mini-tablet (CRMT):

Atazanavir and Ritonavir were complexed with Poloxamer 188 by the solvent evaporation method. Poloxamer 188 was dissolved in methanol, drugs were added to form a homogeneous solution, and then dried under vacuum. For matrix formation, HPMC grades K4M, K15M, and K100M CR Premium in different concentrations (15% and 20%) in formulations M1 to M6 were used. HPMC was mixed with the drug-Poloxamer complex, and further blended with Microcrystalline Cellulose PH 102, Crospovidone XL 10 and Magnesium Stearate. The final blend was compressed into 125 mg mini-tablets using a 6.5 mm punch and was optimized with increased tablet thickness at a reduced compression pressure. The tablet weight and thickness were monitored for their specific parameters. 

In-vitro dissolution study of immediate and controlled release mini-tablets (IRMT and CRMT):

The dissolution test was conducted using USP Type-II paddle apparatus at 50 RPM and maintained at 37°C ± 0.5°C. For IRMT batches L1 to L4, dissolution media was composed of acidic medium (0.1M HCl) with 500 ml at intervals ranging from 5 to 60 min. The CRMT batches M1 to M6 were introduced in 900 ml of sodium acetate buffer (pH 5.5) and samples were withdrawn at intervals of 1 to 12 hours. All samples taken out (5 ml for IRMT and 10 ml for CRMT) were filtered and replaced with the same volume of fresh dissolution media throughout the entire dissolution [13]. Drug release was analyzed spectrophotometrically at 249 nm and 270 nm using the simultaneous estimation method via UV spectrophotometry.

Preparation of pH 5.5 and pH 7 hydro-alcoholic coating solutions:

The coating procedure was done by using the pan coating method with a spray gun for the selected controlled-release mini-tablet (CRMT) formulation which showed the most favorable in-vitro dissolution profile. Sodium citrate was dissolved in purified water while Eudragit L 100-55 (for pH 5.5) and Eudragit S 100 (for pH 7) was dissolved in a mixture of dichloromethane (DCM) and isopropyl alcohol (IPA) at concentrations of 5% or 10%. Quinoline Yellow WS (pH 5.5) and Sunset Yellow Supra (pH 7) were added for coloring. Then, the sodium citrate solution was slowly added into the polymer mixtures under constant stirring, and the pH was controlled at 5.5 or 7 with the help of a pH meter. The prepared coating solutions were applied on CRMT in a pan coater set at an inlet temperature of 30°C, bed temperature of 28°C, and a rotation speed of 8–15 rpm. The final coated tablets were visually inspected for uniformity and evaluated for quality.

Preparation of encapsulated mini-tablets (EMT):

The coated mini-tablets were encapsulated into ‘0’ size capsules. Each capsule has one immediate release mini-tablet (IRMT) and two controlled release mini-tablets (CRMT) coated with pH 5.5 and pH 7 layer. The CRMTs are at the base of the capsule, followed by the IRMT, ensuring uniform arrangement in the capsule. The capsules were securely sealed and completed the encapsulation process.

Drug content evaluation [14]:

Preparation of standard and sample solutions:

Standard solutions were prepared by dissolving 30 mg of Atazanavir and 20 mg of Ritonavir in 100 ml of a methanol: water mixture (1:1) and sonicating the solution. From each stock solution, 10 ml was diluted to 50 ml, and 5 ml of this intermediate solution was further diluted to 50 ml, yielding final concentrations of 30 μg/ml (Atazanavir) and 10 μg/ml (Ritonavir).

For sample solutions, ten encapsulated mini-tablets (EMTs) were crushed, and the average weight was dissolved in 100 ml of methanol: water (1:1) with sonication for 15 minutes. The resulting solution was filtered using a 0.45 μm syringe filter, and 5 ml of the filtrate was diluted to 50 ml in a volumetric flask.

Analysis via simultaneous equation method:

Absorbance at 270 nm and 249 nm was measured, and concentrations of Atazanavir (Ca) and Ritonavir (Cr) were calculated using:

          Ca = A2ay1- A1ay2 / ax2ay1- ax1ay2

          Cr = A1ax2- A2ax1 / ax2ay1-ax1ay2

Where,

A1 and A2 are absorbances of formulation at 270 and 249nm respectively,

Ca and Cr are the concentration of Atazanavir and Ritonavir respectively,

ax1 and ax2 are absorptivities of Atazanavir at 270 and 249nm,

ay1 and ay2 are absorptivities of Ritonavir at 270 and 249nm.

Once the concentrations of Atazanavir (Ca) and Ritonavir (Cr) are determined using the simultaneous equation method, the next step is to calculate the actual amount of each drug in the sample. The drug amount can be calculated with the following formula:

Amount of drug= Concentration Ca or Cr x Dilution factor x Average weight of tablet x Standard concentration (μg/ml)Test weight (mg)

Once the amount of drug is calculated, the percentage of drug content (purity % assay) can then be determined as follows:

% Purity= Amount of drug (mg)Label claim x 100

In-vitro dissolution study of encapsulated mini-tablet (EMT) [15]:

In-vitro dissolution tests for the EMT, IRMT and coated CRMTs were conducted across five separate dissolution runs, with dissolution media adjusted at calculated gastric emptying time intervals. A USP Type-2 (Paddle) apparatus with a paddle rotation speed of 50 RPM and a controlled temperature of 37°C ± 0.5°C was used. For the IRMT sample, 500 ml of 0.1M HCl (8.5 ml HCl to 1000 ml of water) was used as the primary dissolution medium. Samples were withdrawn at specified intervals of 5, 10, 15, 20, 30, 45, 60, and 120 minutes with dissolution medium replaced after each sampling. At the 3rd hour, the pH of the medium was adjusted from 1.2 to 5.5 by gently adding 250 ml of a 6.34 g ammonium phosphate solution without disturbing the dissolved medium content. Additional samples were taken at intervals of 3, 4, and 5 hours, with the medium replaced after each sampling. At the 6th hour, the pH of the medium was further adjusted to 7 by gently adding 250 ml of a 9.5 g ammonium phosphate buffer solution to the existing 750 ml, and subsequent samples were withdrawn at intervals of 6, 7, 8, 9, and every hour up to the 15th hour. Each sample was filtered and analyzed using a UV spectrophotometer at wavelengths of 249 nm and 270 nm. The same dissolution procedure was followed for the 5% pH 5.5 coated CRMT, 10% pH 5.5 coated CRMT, 5% pH 7 coated CRMT, and 10% pH 7 coated CRMT samples.

In-vitro release kinetics studies:

The dissolution profiles of the EMT capsule were analyzed in different kinetic models: Zero-order, First-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell. The best-fit model for each formulation was identified based on the highest regression values of the correlation coefficients, ensuring an accurate drug release mechanism [16, 17].

Stability study:

Stability testing of IRMT and CRMT was performed following ICH guidelines to assess quality variations under environmental factors such as temperature, humidity, and light. Mini-tablets were kept in HDPE containers under accelerated conditions at 40°C ± 2°C, 75% ± 5% RH [18]. They are assessed at predetermined intervals on the physical appearance, hardness, friability, and drug content.

RESULTS:

The main objective of this research was to develop a mini-tablet-filled capsule formulation aimed at reducing the drug dosage while ensuring prolonged drug presence in system. To achieve this, calculated doses of Atazanavir and Ritonavir were incorporated into mini-tablets using different concentrations of individual polymers.  The mini-tablets were filled into a size '0' capsule, selected to be the largest size readily accepted by the humans. Each capsule contained one immediate release mini-tablet, IRMT and two controlled release mini-tablets CRMT, coated with pH dependent polymers targeting release at pH 5.5 and 7 respectively as shown in figure 1. The total dose of drug per capsule was 145 mg of Atazanavir and 55 mg of Ritonavir.