IGF-II-Conjugated Nanocarrier for Brain-Targeted Delivery of p11 Gene for Depression.

Gene therapy involving p11 cDNA has been thought to be a futuristic approach for the effective management of depression as the existing treatment regimen presents many issues regarding late onset of action, patient withdrawal and their side effects. For the effective transfection of p11 gene intracellularly, two cationic lipids based on phospholipid DOPE conjugated to basic amino acids histidine and arginine were synthesised, used for liposome formulation and evaluated for their ability as gene delivery vectors. They were further converted using IGF-II mAb into immunoliposomes for CNS targeting and mAb conjugation to liposomes were characterised by SDS-PAGE. They were further analysed by in vitro characterisation studies that include erythrocyte aggregation study, electrolyte-induced study, heparin compatibility study and serum stability studies. SHSY5Y cells were used for conducting cytotoxicity of synthesised lipids and live imaging of cell uptake for 25 min. Finally, the brain distribution studies and western blot were carried out in animals to evaluate them for their BBB permeation ability and effects on p11 protein which is believed to be a culprit. These formulated liposomes from synthesised lipids offer a promising approach for the treatment of depression.

Liposomes encapsulating native and cyclodextrin enclosed paclitaxel: Enhanced loading efficiency and its pharmacokinetic evaluation.

Combination strategy involving cyclodextrin (CD) complexation and liposomal system was investigated for Paclitaxel (PTX) to improve loading. Complexation was done using 2,6-di-O-methylbetacyclodextrin (DMßCD). Sterically stabilized double loaded PEGylated liposomes (DLPLs) containing PTX and PTX-DMßCD complex were prepared by thin film hydration. Physicochemical characterization of complex and prepared DLPLs was carried out. Cytotoxic potential, hemolytic potential and pharmacokinetics of DLPLs were tested in comparison to Taxol®. Aqueous solubility of PTX increased by almost 3 × 104 folds due to complexation with DMßCD as compared to pure drug solubility. Liposomal system was found to have 162.8 ± 4.1 nm size, zeta potential of -5.6 ± 0.14 mV and 2-fold increase in drug loading to 5.8 mol % for PTX due to double loading. DLPLs had low hemolytic potential and higher cytotoxicity on SKOV3 cells with improvement in IC50 value by 4.2 folds as compared to Taxol® at 48 h. The anti-angiogenic potential of DLPLs was confirmed by 1.33 folds lesser wound recovery in SKOV3 cells compared to Taxol®. In-vivo pharmacokinetic evaluation of DLPLs in rats substantiates improvement in circulation time, higher plasma concentration and decreased clearance rate compared to Taxol®. An efficacious system with improved loading and pharmacokinetics was formulated as potential alternative for currently marketed PTX formulation.

Quality by Design Empowered Development and Optimisation of Time-Controlled Pulsatile Release Platform Formulation Employing Compression Coating Technology.

The research was envisaged for development of time-controlled pulsatile release (PR) platform formulation to facilitate management of early morning chronological attacks. The development was started using prednisone as a model drug wherein core tablets were prepared using direct compression method and subsequently compression-coated with ethylcellulose (EC)-hydroxypropyl methylcellulose (HPMC) excipient blend. Initially, quality target product profile was established and risk assessment was performed using failure mode and effect analysis. In an endeavour to accomplish the objective, central composite design was employed as a design of experiment (DoE) tool. Optimised compression-coated tablet (CCT) exhibited 4-6 h lag time followed by burst release profile under variegated dissolution conditions viz. multi-media, change in apparatus/agitation and biorelevant media. Afterwards, five different drugs, i.e. methylprednisolone, diclofenac sodium, diltiazem hydrochloride, nifedipine and lornoxicam, were one-by-one incorporated into the optimised prednisone formula with replacement of former drug. Change in drug precipitated the issues like poor solubility and flow property which were respectively resolved through formulation of solid dispersion and preparation of active pharmaceutical ingredient (API) granules. Albeit, all drug CCTs exhibited desired release profile similar to prednisone CCTs. In nutshell, tour de force of research epitomised the objective of incorporating diverse drug molecules and penultimately obtaining robust release profile at varying dissolution conditions.

Formulation Development, Process Optimization, and In Vitro Characterization of Spray-Dried Lansoprazole Enteric Microparticles.

This research focuses on the development of enteric microparticles of lansoprazole in a single step by employing the spray drying technique and studies the effects of variegated formulation/process variables on entrapment efficiency and in vitro gastric resistance. Preliminary trials were undertaken to optimize the type of Eudragit and its various levels. Further trials included the incorporation of plasticizer triethyl citrate and combinations of other polymers with Eudragit S 100. Finally, various process parameters were varied to investigate their effects on microparticle properties. The results revealed Eudragit S 100 as the paramount polymer giving the highest gastric resistance in comparison to Eudragit L 100-55 and L 100 due to its higher pH threshold and its polymeric backbone. Incorporation of plasticizer not only influenced entrapment efficiency, but diminished gastric resistance severely. On the contrary, polymeric combinations reduced entrapment efficiency for both sodium alginate and glyceryl behenate, but significantly influenced gastric resistance for only sodium alginate and not for glyceryl behenate. The optimized process parameters were comprised of an inlet temperature of 150°C, atomizing air pressure of 2 kg/cm(2), feed solution concentration of 6% w/w, feed solution spray rate of 3 ml/min, and aspirator volume of 90%. The SEM analysis revealed smooth and spherical shape morphologies. The DSC and PXRD study divulged the amorphous nature of the drug. Regarding stability, the product was found to be stable under 3 months of accelerated and long-term stability conditions as per ICH Q1A(R2) guidelines. Thus, the technique offers a simple means to generate polymeric enteric microparticles that are ready to formulate and can be directly filled into hard gelatin capsules.

Role of antibodies in diagnosis and treatment of ovarian cancer: Basic approach and clinical status.

Ovarian cancer is the second most leading gynecological cancer after endometrial cancer in women. Chemotherapy and cyto-reductive surgery are currently the mainstays for treatment of ovarian cancer in early stages. However, the overall 5years of survival rate in advanced stage is just 20-30%. The main reasons behind therapeutic failures and a low survival rate are challenges in early diagnosis, frequent recurrence, chemo-resistance development and lack of targeting. Antibodies have evolved as a rationale therapeutic approach to overcome these hurdles and to engender significant clinical applications. Detection of cancer associated antibodies and radiolabeled antibody conjugates are forming the basis for early diagnosis of ovarian cancer. Besides this, antibodies when given alone act as an anti-angiogenic agent or utilize the protective role of immune system against ovarian cancer. This high specificity and selectivity of action is also accompanied by development of tumor antigen-specific memory T cells, which can prevent cancer recurrence. In addition, when given in combination with chemotherapy, antibodies have turned out to sensitize chemo-resistant tumors. Antibodies are also playing cardinal role in design of highly potent class of targeted therapies, such as antibody-drug conjugates and clinically viable tumor targeted drug nanocarriers. This article aims to explore the fundamentals in development of antibody based therapeutics and multitude ways of clinical applications in diagnosis and treatment of ovarian cancer. Focus is given on the recent advances made in preclinical and clinical settings with an overview to unmet challenges so as to develop better immunotherapeutics in future.

Preparation and characterization of dipyridamole solid dispersions for stabilization of supersaturation: effect of precipitation inhibitors type and molecular weight.

Dipyridamole (DPL) is a weakly basic BCS class II drug which precipitates upon entering into intestine leading to pH dependant and variable absorption. Thus, research envisaged focuses on developing formulations that maintain supersaturation following upon acid to neutral pH transition. In an endeavor to accomplish the objective, solid dispersion (SD) with hydroxypropylmethyl cellulose (HPMC) and polyvinylpyrrolidone (PVP) was prepared by a quench cooling method. The three molecular weight grades of HPMC (HPMC E5, HPMC E15 and HPMC E50) and two molecular weight grades of PVP (PVP K30 and PVP K90) were investigated to observe effect of increasing molecular weight on stabilizing DPL supersaturated solutions. Equilibrium solubility studies revealed increase in solubility with both HPMC and PVP with greater benefit from HPMC. In vitro supersaturated dissolution results demonstrated that HPMC formulations provided greater degree and extent of supersaturation as compared to PVP formulations. The formulation with HPMC E50 provided maximum stabilization to supersaturation upon acid to neutral pH transition. Moreover, the effect of increase in molecular weight was more pronounced in HPMC rather than PVP. Stronger interactions were observed for DPL with HPMC, while no interaction was observed with PVP which was evident from Fourier transform infra-red studies. Differential scanning calorimetry and powder X-ray diffraction studies revealed the amorphous state of DPL in SD.

Investigating effects of hydroxypropyl methylcellulose (HPMC) molecular weight grades on lag time of press-coated ethylcellulose tablets.

The research undertaken exemplifies the effects of hydroxypropyl methylcellulose (HPMC) molecular weight (MW) grades of on lag time of press-coated ethylcellulose (EC) tablets. The formulation comprised an immediate release core (containing prednisone as a model drug) surrounded by compression coating with variegated EC-HPMC blends. Five selected HPMC grades (E5, E15, E50, K100LV and K4M) were explored at three different concentrations (10% w/w, 20% w/w and 30% w/w in outer coat) to understand their effects on lag time and drug release. In vitro drug release testing demonstrated that, with increase in concentration of E5 and E15, up to 30% w/w, the mean lag time decreased progressively; whereas with remaining grades, the mean lag time initially decreased up to 20% w/w level and thereafter increased for 30% w/w level. Importantly, with increase in HPMC concentration in the outer coat, the variability in lag time (%RSD; n = 6) was decreased for each of E5, E15 and E50, whereas increased for K100LV and K4M. In general, the variability in lag time was increased with increase in HPMC MW at studied concentration levels. Markedly, tablets with 30% w/w K4M in outer coat exhibited slight premature release (before the rupture of outer coat) along with high variability in lag time. Overall, the study concluded that low MW HPMCs (E5, E15 and E50) were found rather efficient than higher MW HPMCs for developing robust EC-based press-coated pulsatile release formulations where precise lag time followed by sharp burst release is desired.

Investigating critical effects of variegated lubricants, glidants and hydrophilic additives on lag time of press coated ethylcellulose tablets.

The research envisaged focuses on vital impacts of variegated lubricants, glidants and hydrophilic additives on lag time of press coated ethylcellulose (EC) tablets using prednisone as a model drug. Several lubricants and glidants such as magnesium stearate, colloidal SiO2, sodium stearyl fumarate, talc, stearic acid, polyethylene glycol (6000) and glyceryl behenate were investigated to understand their effects on lag time by changing their concentrations in outer coat. Further, the effects of hydrophilic additives on lag time were examined for hydroxypropylmethylcellulose (E5), hydroxypropylcellulose (EF and SSL), povidone (K30), copovidone, polyethylene glycol (4000), lactose and mannitol. In vitro drug release testing revealed that each selected lubricant/glidant, if present even at concentration of 0.25% w/w, significantly reduced the lag time of press coated tablets. Specifically, colloidal SiO2 and/or magnesium stearate were detrimental while other lubricants/glidants were relatively less injurious. Among hydrophilic additives, freely water soluble fillers had utmost influence in lag time, whereas, comparatively less impact was observed with polymeric binders. Concisely, glidant and lubricant should be chosen to have minimal impact on lag time and further judicious selection of hydrophilic additives should be exercised for modulating lag time of pulsatile release formulations.

Myriad Molecules to Overcome Efflux Drug Transporters and Drug-Metabolizing Enzymes: A Journey from Synthetic to Natural.

A vast range of prescribed drugs suffers from low and variable bioavailability mainly because of metabolism and permeation complications. This issue of bioavailability is a key problem that has been ongoing for many years. Various tactics have been introduced that have been quite beneficial for improving the bioavailability of poorly bioavailable drugs. Some of these tactics are targeted on cytochrome-P450 (CYP) enzymes and the permeability-glycoprotein (P-gp) efflux pump. Strategies include small-molecule inhibitors, novel drug-delivery systems, pharmaceutical synthetic excipients, and natural bioenhancers. This review discusses the role of synthetic excipients and natural bioenhancers in potentiating the activity of poorly bioavailable drugs, including their pharmacological background and their future applicability to health care. The molecules of synthetic origin such as Gelucire and those of natural origin such as quercetin and silibinin can provide noteworthy benefits to patients and the health care system by helping to reduce dosing and drug side effects.

Inhalable liposomal dry powder of gemcitabine-HCl: Formulation, in vitro characterization and in vivo studies.

Pulmonary drug delivery system facilitates local instillation of anticancer drugs to lungs which has proven to be pioneering approach for treatment of lung cancer. This approach led the groundwork for delivering liposomal formulation directly to lungs. Gemcitabine-HCl is currently considered as most effective drug for management of lung cancer. However, its application is limited owing to its metabolism by enzymes present in plasma resulting in reduced efficacy and higher toxicity. In present study, lyophilisation technique was used to convert liposomes into dry powder inhaler, which was formulated using emulsification solvent evaporation technique. The physicochemical properties including size, morphology, entrapment efficiency, loading efficiency etc. of formulated liposomes were evaluated. The prepared liposomal DPI (LDPI) formulations were then examined for solid state characteristics and aerosol performance using cascade impactor. From all the formulations prepared, the LDPI formulated using trehalose as cryoprotectant presented required properties along with desirable deposition pattern. Finally, the optimized formulation was selected for in vitro cell line studies; in vivo studies and stability study. This formulated inhalable particles offers a promising approach for the management of lung cancer through regional chemotherapy.

A newfangled study using risk silhouette and uncertainty approximation for quantification of acyclovir in diverse formulation.

Risk assessment and uncertainty approximation are two major and important parameters that need to be adopted for the development of pharmaceutical process to ensure reliable results. Additionally, there is a need to switch from the traditional method validation checklist to provide a high level of assurance of method reliability to measure quality attribute of a drug product. In the present work, evaluation of risk profile, combined standard uncertainty and expanded uncertainty in the analysis of acyclovir were studied. Uncertainty was calculated using cause-effect approach, and to make it more accurately applicable a method was validated in our laboratory as per the ICH guidelines. While assessing the results of validation, the calibration model was justified by the lack of fit and Levene׳s test. Risk profile represents the future applications of this method. In uncertainty the major contribution is due to sample concentration and mass. This work demonstrates the application of theoretical concepts of calibration model tests, relative bias, risk profile and uncertainty in routine methods used for analysis in pharmaceutical field.

Dissolution criticality in developing solid oral formulations: from inception to perception.

Currently, dissolution testing has become a vital tool for accessing product performance, especially in the hierarchy of solid oral dosage forms. With advances in complicated, expensive, and sophisticated analytical instruments, characterization of formulations has become easier, but simple dissolution assembly is gradually gaining momentum from industrial environs as well as regulatory agencies. As such, simple dissolution testing involves many complexities which must be properly understood to reach correct conclusions. The appropriate selection of multiple parameters (e.g., apparatus, medium, agitation, etc.) involved in dissolution testing and understanding their impact on analysis require thorough subject knowledge. In the words of regulatory provisions, in vitro dissolution testing can become a surrogate for expensive and tedious bioequivalence studies in special cases (i.e., when a biowaiver is recommended). As a consequence, reduced human testing as well as lower product development cost ultimately benefit patients and society. Therefore, the dissolution science has recently become one of the keys for success for formulation scientists, especially generic manufacturers. While designing dissolution methodologies, generic manufacturers need to follow the respective regulatory guidelines at the product development stage; concomitant data are required for the approval process. This comprehensive review is an earnest attempt to acquaint readers with the history, contemporary practices, and relevant issues regarding dissolution which may become a guiding tool for overcoming challenges and opening better prospects in product development.

Risk based approach for design and optimization of stomach specific delivery of rifampicin.

The research envisaged focuses on risk management approach for better recognizing the risks, ways to mitigate them and propose a control strategy for the development of rifampicin gastroretentive tablets. Risk assessment using failure mode and effects analysis (FMEA) was done to depict the effects of specific failure modes related to respective formulation/process variable. A Box-Behnken design was used to investigate the effect of amount of sodium bicarbonate (X1), pore former HPMC (X2) and glyceryl behenate (X3) on percent drug release at 1st hour (Q1), 4th hour (Q4), 8th hour (Q8) and floating lag time (min). Main effects and interaction plots were generated to study effects of variables. Selection of the optimized formulation was done using desirability function and overlay contour plots. The optimized formulation exhibited Q1 of 20.9%, Q4 of 59.1%, Q8 of 94.8% and floating lag time of 4.0 min. Akaike information criteria and Model selection criteria revealed that the model was best described by Korsmeyer-Peppas power law. The residual plots demonstrated no existence of non-normality, skewness or outliers. The composite desirability for optimized formulation computed using equations and software were 0.84 and 0.86 respectively. FTIR, DSC and PXRD studies ruled out drug polymer interaction due to thermal treatment.

Recent patents and advances on anti - tuberculosis drug delivery and formulations.

Tuberculosis has remained, unambiguously, a significant health care problem since long times, particularly in developing countries. The endeavoring battle against multi drug resistant TB, multiple dosing, their prominent side effects and bioavailability hiccups related to fixed dose combinations has undeniably become a Herculean task indicating rigorous research requirement in anti TB drug therapy. In view of the fact that patenting a drug molecule, a drug delivery system or a formulation has been very fruitful for the growth and sustainment of pharmaceutical industry, a meticulous review of recent developments, providing a balanced view on merits/demerits, will facilitate researchers to update themselves, thereby focusing their research in more relevant areas to furnish desired quality traits. This article reviews the present scenario in terms of drug delivery approaches for TB chemotherapy. The review encompasses and summarizes recent patents and advances on variegated facets of dosage forms, together with from conventional solid oral to novel controlled release oral formulations and additionally alternative weapons for anti TB drug delivery. A critical review of multidisciplinary approaches to boost anti TB therapy may facilitate the scientists to resolve existing technological gaps.

Design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clindamycin phosphate.

To develop more effective treatment for bacterial vaginosis, bioadhesive film formulations of clindamycin phosphate (CL) for vaginal delivery were studied. The bioadhesive films were prepared by solvent evaporation method. A 3(2) full factorial design was utilized for optimization of the effect of independent variables such as amount of hydroxypropyl cellulose (X1), amount of xanthan gum (X2) on mechanical properties, and % drug retained on vaginal mucosa. The films were evaluated for various aesthetic and physicodynamic properties. Batch F(86) showed highest overall desirability of 0.99. Batch F86 was considered optimal composition for a novel bioadhesive vaginal formulation, as they have good peelability, high % elongation at break, moderate tensile strength, and retained on vaginal mucosa up to 8 h. Also, films were non-cytotoxic as indicated by negligible decrease in cell viability. Our study may provide a potential vaginal delivery system of CL against bacterial vaginosis.

Palatable reconstitutable dry suspension of artemether for flexible pediatric dosing using cyclodextrin inclusion complexation.

The present research was conducted to investigate the inclusion complexation of artemether (ARM) with beta-cyclodextrin (CD) with the aim of masking the bitterness along with improving the drug release and preparing a stable palatable formulation of ARM especially for pediatrics. A physical mixture and kneaded system were prepared to study the inclusion complexation. Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD) and scanning electron microscopy (SEM) were performed to identify the physicochemical interaction between drug and carrier, hence its effect on drug release. Reconstitutable dry suspension was evaluated for angle of repose, sedimentation characterization and pH. In vitro drug release studies for physical mixture and kneaded system were performed at pH, 1.2 and 6.8. Bitterness score was evaluated using gustatory sensation test. The FTIR, DSC and XRPD studies indicated inclusion complexation in physical mixture and kneaded system. In addition, physical mixture and kneaded system exhibited improved drug release at pH, 1.2 and 6.8. To formulate palatable reconstitutable dry suspension of ARM, the 1:20M physical mixture was selected based on bitterness score. Reconstitutable dry suspension prepared using physical mixture (DS4), showed complete bitter taste masking, good flowability and ease of redispersibility. Taste evaluation of reconstitutable dry suspension in human volunteers rated tasteless with a score of 0 to DS4 and 3 to DS5 (reconstitutable dry suspension prepared using pure ARM). This conclusively demonstrated a stable and palatable reconstitutable dry suspension of ARM using CD inclusion complexation for flexible pediatric dosing.

Development and evaluation of sustained release gastroretentive minimatrices for effective treatment of H. pylori infection.

In the present work, sustained release gastroretentive minimatrices of amoxicillin have been designed and optimized using central composite design. Effect of amount of xanthan gum, rate controlling polymers (HPMC K100M CR/PEO coagulant (1:1)), carbopol 974P, and gas generating couple (sodium bicarbonate/citric acid (3:1)) was studied on dependent (response) variables, i.e., buoyancy lag time, drug release at 1 h, time required for 95% drug release, swelling index, and bioadhesive strength. Minimatrices were prepared by non aqueous granulation method using solution of PVP K30 in isopropyl alcohol. All the formulations were found to contain 99.2% to 100.9% of amoxicillin per minimatrix. Optimum formulation (Formulation number AGT09) containing high level of the independent variables was having buoyancy lag time of 7 min and drug release at 1 h was 32.5%. It required 9.39 h for 95% drug release while swelling index and bioadhesive strength were 341 and 17.9 dyn/cm(2), respectively. This formulation was said to be optimum because it has minimum buoyancy lag time, requires maximum time for 95% drug release, and has higher bioadhesive capabilities. In vitro results of an optimized formulation indicate its sustained drug release and gastric retention capability, which may be very useful for effective treatment of H. pylori infection.

Influence of chitosan crosslinking on bitterness of mefloquine hydrochloride microparticles using central composite design.

The present work examines the influence of various process and product parameters on mefloquine hydrochloride (MFL) entrapped in crosslinked chitosan microparticles for masking the bitterness. A central composite design (CCD) was employed to investigate the effect of three process and product variables, namely amount of MFL, chitosan and sodium hydroxide (crosslinking agent) on the incorporation efficiency, particle size, drug release at pH 6.8 and bitterness score. The microparticles were prepared by ionotropic gelation method, with a hardening time of 60 min. The optimum condition for process and product variables was evaluated using desirability function. The model is further cross validated for bias. The optimized microparticles were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. Bitterness score was evaluated by human gustatory sensation test. Multiple linear regression analysis revealed that the crosslinking of chitosan significantly affects incorporation efficiency, particle size, drug release and bitterness score. The bitterness score was decreased to zero compared to 3+ of pure MFL. It can be inferred that the proposed methodology can be used to prepare MFL microparticles for bitter taste masking.

Design and optimization of artemether microparticles for bitter taste masking.

The objective of the present investigation was to reduce the bitterness of artemether (ARM). Microparticles were prepared by the coacervation method using Eudragit E 100 (EE) as polymer and sodium hydroxide solution as nonsolvent for the polymer. A 32 full factorial design was used for optimization wherein the amount of drug (A) and polymer (B) were selected as independent variables and the bitterness score, particle size and drug release at pH, 1.2 and 6.8 were selected as dependent variables. Optimization was carried out using the desirability function. The optimized microparticles batch was characterized by FTIR and DSC. Multiple linear regression analysis revealed that reduced bitterness of ARM can be obtained by controlling the drug release of microparticles at pH 6.8 and increasing the amount of EE. The increase in the amount of polymer leads to reduction in drug release from microparticles at pH > 5 due to its insolubility and thus reduces bitterness. However, the increase in the amount of polymer results in improved dissolution, suggesting improved availability of ARM in stomach. Optimized microparticles prepared using 0.04 g of ARM and 15 mL of 1% (m/V) solution of EE showed complete bitter taste masking with improved drug release at pH 1.2.

Formulation and evaluation of primaquine phosphate taste-masked rapidly disintegrating tablet.

This work investigates the complete bitter-taste-masking of primaquine phosphate (PRM) using a solid dispersion with mono ammonium glycyrrhyzinate pentahydrate (GLY). This work also describes the preparation of rapidly disintegrating tablets (RDTs) of PRM by a direct compression method using superdisintegrant, croscarmellose sodium. A solid dispersion was prepared by the solvent evaporation method. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were performed to identify the physicochemical interaction between drug and carrier, hence its effect on dissolution. In-vitro drug release studies were performed for RDTs at both pH 1.2 and 6.8. Bitterness score was evaluated using a human gustatory sensation test. FTIR spectroscopy and DSC showed no interaction of PRM in GLY solid dispersion. RDTs prepared from solid dispersion showed complete bitter-taste-masking of PRM. RDTs containing solid dispersion exhibited a better dissolution profile, at both pH 1.2 and 6.8, than pure PRM. Thus, the solid dispersion technique can be successfully used for complete bitter taste masking of PRM.