HSPB4/CRYAA Protect Photoreceptors during Retinal Detachment in Part through FAIM2 Regulation.

Our previous study discussed crystallin family induction in an experimental rat model of retinal detachment. Therefore, we attempted to evaluate the role of α-crystallin in photoreceptor survival in an experimental model of retinal detachment, as well as its association with the intrinsically neuroprotective protein Fas-apoptotic inhibitory molecule 2 (FAIM2). Separation of retina and RPE was induced in rat and mouse eyes by subretinal injection of hyaluronic acid. Retinas were subsequently analyzed for the presence αA-crystallin (HSPB4) and αB-crystallin (HSPB5) proteins using immunohistochemistry and immunoblotting. Photoreceptor death was analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining and cell counts. The 661W cells subjected to FasL were used as a cell model of photoreceptor degeneration to assess the mechanisms of the protective effect of αA-crystallin and its dependence on its phosphorylation on T148. We further evaluated the interaction between FAIM2 and αA-crystallin using a co-immunoprecipitation assay. Our results showed that α-crystallin protein levels were rapidly induced in response to retinal detachment, with αA-crystallin playing a particularly important role in protecting photoreceptors during retinal detachment. Our data also show that the photoreceptor intrinsically neuroprotective protein FAIM2 is induced and interacts with α-crystallins following retinal detachment. Mechanistically, our work also demonstrated that the phosphorylation of αA-crystallin is important for the interaction of αA-crystallin with FAIM2 and their neuroprotective effect. Thus, αA-crystallin is involved in the regulation of photoreceptor survival during retinal detachment, playing a key role in the stabilization of FAIM2, serving as an important modulator of photoreceptor cell survival under chronic stress conditions.

Ocular manifestations of central insulin resistance.

Central insulin resistance, the diminished cellular sensitivity to insulin in the brain, has been implicated in diabetes mellitus, Alzheimer's disease and other neurological disorders. However, whether and how central insulin resistance plays a role in the eye remains unclear. Here, we performed intracerebroventricular injection of S961, a potent and specific blocker of insulin receptor in adult Wistar rats to test if central insulin resistance leads to pathological changes in ocular structures. 80 mg of S961 was stereotaxically injected into the lateral ventricle of the experimental group twice at 7 days apart, whereas buffer solution was injected to the sham control group. Blood samples, intraocular pressure, trabecular meshwork morphology, ciliary body markers, retinal and optic nerve integrity, and whole genome expression patterns were then evaluated. While neither blood glucose nor serum insulin level was significantly altered in the experimental or control group, we found that injection of S961 but not buffer solution significantly increased intraocular pressure at 14 and 24 days after first injection, along with reduced porosity and aquaporin 4 expression in the trabecular meshwork, and increased tumor necrosis factor α and aquaporin 4 expression in the ciliary body. In the retina, cell density and insulin receptor expression decreased in the retinal ganglion cell layer upon S961 injection. Fundus photography revealed peripapillary atrophy with vascular dysregulation in the experimental group. These retinal changes were accompanied by upregulation of pro-inflammatory and pro-apoptotic genes, downregulation of anti-inflammatory, anti-apoptotic, and neurotrophic genes, as well as dysregulation of genes involved in insulin signaling. Optic nerve histology indicated microglial activation and changes in the expression of glial fibrillary acidic protein, tumor necrosis factor α, and aquaporin 4. Molecular pathway architecture of the retina revealed the three most significant pathways involved being inflammation/cell stress, insulin signaling, and extracellular matrix regulation relevant to neurodegeneration. There was also a multimodal crosstalk between insulin signaling derangement and inflammation-related genes. Taken together, our results indicate that blocking insulin receptor signaling in the central nervous system can lead to trabecular meshwork and ciliary body dysfunction, intraocular pressure elevation, as well as inflammation, glial activation, and apoptosis in the retina and optic nerve. Given that central insulin resistance may lead to neurodegenerative phenotype in the visual system, targeting insulin signaling may hold promise for vision disorders involving the retina and optic nerve.

αA-Crystallin Mediated Neuroprotection in the Retinal Neurons Is Independent of Protein Kinase B.

Phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway mediates pro-survival function in neurons. In the retina, PI3K/AKT/mTOR signaling pathway is related to the early pathogenesis of diabetic retinopathy. Signaling molecules in the membrane-initiated signaling pathway exhibiting neuroprotective function interacts with the PI3K/Akt pathway as an important survival pathway. Molecular chaperone α-crystallins are known to potentially interact and/or regulate various pro-survival and pro-apoptotic proteins to regulate cell survival. Among these demonstrated mechanisms, they are well-reported to regulate and inhibit apoptosis by interacting and sequestrating the proapoptotic proteins such as Bax and Bcl-Xs. We studied the importance of metabolic stress-induced enhanced Akt signaling and αA-crystallin interdependence for exhibiting neuroprotection in metabolically challenged retinal neurons. For the first time, this study has revealed that αA-crystallin and activated Akt are significantly neuroprotective in the stressed retinal neurons, independent of each other. Furthermore, the study also highlighted that significant inhibition of the PI3K-Akt pathway does not alter the neuroprotective ability of αA-crystallin in stressed retinal neurons. Interestingly, our study also demonstrated that in the absence of Akt activation, αA-crystallin inhibits the translocation of Bax in the mitochondria during metabolic stress, and this function is regulated by the phosphorylation of αA-crystallin on residue 148.

Evidence for Paracrine Protective Role of Exogenous αA-Crystallin in Retinal Ganglion Cells.

Expression and secretion of neurotrophic factors have long been known as a key mechanism of neuroglial interaction in the central nervous system. In addition, several other intrinsic neuroprotective pathways have been described, including those involving small heat shock proteins such as α-crystallins. While initially considered as a purely intracellular mechanism, both αA-crystallins and αB-crystallins have been recently reported to be secreted by glial cells. While an anti-apoptotic effect of such secreted αA-crystallin has been suggested, its regulation and protective potential remain unclear. We recently identified residue threonine 148 (T148) and its phosphorylation as a critical regulator of αA-crystallin intrinsic neuroprotective function. In the current study, we explored how mutation of this residue affected αA-crystallin chaperone function, secretion, and paracrine protective function using primary glial and neuronal cells. After demonstrating the paracrine protective effect of αA-crystallins secreted by primary Müller glial cells (MGCs), we purified and characterized recombinant αA-crystallin proteins mutated on the T148 regulatory residue. Characterization of the biochemical properties of these mutants revealed an increased chaperone activity of the phosphomimetic T148D mutant. Consistent with this observation, we also show that exogeneous supplementation of the phosphomimetic T148D mutant protein protected primary retinal neurons from metabolic stress despite similar cellular uptake. In contrast, the nonphosphorylatable mutant was completely ineffective. Altogether, our study demonstrates the paracrine role of αA-crystallin in the central nervous system as well as the therapeutic potential of functionally enhanced αA-crystallin recombinant proteins to prevent metabolic-stress induced neurodegeneration.

HspB4/αA-Crystallin Modulates Neuroinflammation in the Retina via the Stress-Specific Inflammatory Pathways.

PURPOSE: We have previously demonstrated that HspB4/αA-crystallin, a molecular chaperone, plays an important intrinsic neuroprotective role during diabetes, by its phosphorylation on residue 148. We also reported that HspB4/αA-crystallin is highly expressed by glial cells. There is a growing interest in the potential causative role of low-grade inflammation in diabetic retinopathy pathophysiology and retinal Müller glial cells' (MGCs') participation in the inflammatory response. MGCs indeed play a central role in retinal homeostasis via secreting various cytokines and other mediators. Hence, this study was carried out to delineate and understand the regulatory function of HspB4/αA-crystallin in the inflammatory response associated with metabolic stresses. METHODS: Primary MGCs were isolated from knockout HspB4/αA-crystallin mice. These primary cells were then transfected with plasmids encoding either wild-type (WT), phosphomimetic (T148D), or non-phosphorylatable mutants (T148A) of HspB4/αA-crystallin. The cells were exposed to multiple metabolic stresses including serum starvation (SS) or high glucose with TNF-alpha (HG + T) before being further evaluated for the expression of inflammatory markers by qPCR. The total protein expression along with subcellular localization of NF-kB and the NLRP3 component was assessed by Western blot. RESULTS: Elevated levels of IL-6, IL-1ß, MCP-1, and IL-18 in SS were significantly diminished in MGCs overexpressing WT and further in T148D as compared to EV. The HG + T-induced increase in these inflammatory markers was also dampened by WT and even more significantly by T148D overexpression, whereas T148A was ineffective in either stress. Further analysis revealed that overexpression of WT or the T148D, also led to a significant reduction of Nlrp3, Asc, and caspase-1 transcript expression in serum-deprived MGCs and nearly abolished the NF-kB induction in HG + T diabetes-like stress. This mechanistic effect was further evaluated at the protein level and confirmed the stress-dependent regulation of NLRP3 and NF-kB by αA-crystallin. CONCLUSIONS: The data gathered in this study demonstrate the central regulatory role of HspB4/αA-crystallin and its modulation by phosphorylation on T148 in retinal MGCs. For the first time, this study demonstrates that HspB4/αA-crystallin can dampen the stress-induced expression of pro-inflammatory cytokines through the modulation of multiple key inflammatory pathways, therefore, suggesting its potential as a therapeutic target for the modulation of chronic neuroinflammation.

Peripheral blood-derived monocytes show neuronal properties and integration in immune-deficient rd1 mouse model upon phenotypic differentiation and induction with retinal growth factors.

BACKGROUND: Cell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. In the current study, we aimed to assess if peripheral blood-derived monocytes which are highly abundant and accessible could be utilized as a potential candidate for phenotypic differentiation into neuron-like cells. METHODS: The peripheral blood-derived monocytes were reconditioned phenotypically using extrinsic growth factors to induce pluripotency and proliferation. The reconditioned monocytes (RM) were further incubated with a cocktail of growth factors involved in retinal development and growth to induce retinal neuron-like properties. These cells, termed as retinal neuron-like cells (RNLCs) were characterized for their morphological, molecular and functional behaviour in vitro and in vivo. RESULTS: The monocytes de-differentiated in vitro and acquired pluripotency with the expression of prominent stem cell markers. Treatment of RM with retinal growth factors led to an upregulation of neuronal and retinal lineage markers and downregulation of myeloid markers. These cells show morphological alterations resembling retinal neuron-like cells and expressed photoreceptor (PR) markers. The induced RNLCs also exhibited relative membrane potential change upon light exposure suggesting that they have gained some neuronal characteristics. Further studies showed that RNLCs could also integrate in an immune-deficient retinitis pigmentosa mouse model NOD.SCID-rd1 upon sub-retinal transplantation. The RNLCs engrafted in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the RP afflicted retina. Mice transplanted with RNLCs showed improvement in depth perception, exploratory behaviour and the optokinetic response. CONCLUSIONS: This proof-of-concept study demonstrates that reconditioned monocytes can be induced to acquire retinal neuron-like properties through differentiation using a defined growth media and can be a potential candidate for cell therapy-based interventions and disease modelling for ocular diseases.

Evaluation of the possibility of selective modulation of retinal glucose transporters in diabetic complications: An experimental study.

PURPOSE: To identify the possibility of modulating retinal glucose transporters in diabetic conditions to prevent retinal complications of diabetic retinopathy. MATERIALS AND METHODS: In silico and in vitro binding assays were performed to assess the effect of genistein and positive controls (pioglitazone and estradiol) on nuclear receptor estrogen receptor beta and peroxisome proliferator-activated receptor gamma (PPARγ). In vivo effects of compounds were tested on diabetic rats. Structural and functional analysis of retina was performed at 28th day followed by gene expression analysis of glucose transporters and nuclear receptors. Pioglitazone and genistein levels were analyzed by liquid chromatography with tandem mass spectrometry. RESULTS: Genistein showed equi-affinity toward PPARγ in in silico experiments contrary to in vitro findings. In multidose study, their therapeutic effects were observed by analyzing the retinal function. Retinal gene expression studies revealed that both test agents significantly up regulated PPARγ, GLUT4, and down regulated GLUT1. Genistein showed significant up regulation of GLUT4 and down regulation of GLUT1 as compared to PGZ which has been well correlated with the Electroretinography (ERG) outcome. CONCLUSION: This study showed the possibility of selective upregulation of GLUT4 (independent of PPARγ activation) in the retina of diabetic rats using genistein. Selective modulation of retinal glucose transporters as therapeutic target in ocular diabetic complications can be possibly explored.

Ocular kinetics and safety of intravitreally injected angiotensin converting enzyme inhibitor lisinopril.

BACKGROUND AND OBJECTIVES: The study investigated the intravitreal safety and vitreous disposition of lisinopril, an angiotensin converting enzyme inhibitor in rabbits for its projected use in retinopathy. METHODS: For the safety study, following the baseline ERG recording and fundus photography, 40 µg/50 µl of lisinopril sterile injection was injected unilaterally in the rabbit eyes (n = 4), where other eye served as a control. The electroretinogram and fundus images were obtained at 24, 48, 72 and 168 h following the intravitreal injection. For pharmacokinetics evaluation of the lisinopril, one eye of each rabbit (n = 4) received an intravitreal injection of lisinopril (40 µg/50 µl). The concentration of lisinopril in the ocular tissues, humours, plasma, lung, kidney and liver were measured through ESI-LC-MS/MS. RESULTS: Upon the electroretinography studies, no significant difference was observed in the ERG pattern in the lisinopril injected eye when compared to the baseline of the respective animals till the 7th day of the study. In the fundus imaging, no morphological changes were observed in the retina of the animal. The concentration of the lisinopril was found to be above to the IC50 in the retina-choroid till 36 h. The concentration found in the plasma and body tissues were many folds less than the IC50 of the lisinopril. CONCLUSIONS: Intravitreal injection of 40 µg/50 µl of lisinopril found to be safe in the rabbit eye as evidenced by the electroretinography and fundus imaging studies. The average half-life of lisinopril is 12.6 h and the above-mentioned dose able to sustain its IC50 value till the 36 h.

An experimental study to evaluate safety/toxicity of intravitreal natalizumab.

PURPOSE: The purpose of this prospective experimental study was to evaluate the safety/toxicity of α4ß1 integrin blockade in rabbit retina using its monoclonal antibody (Natalizumab). METHODS: Twelve New Zealand albino rabbits were divided into three groups (n = 4). Unilateral intravitreal injections of three different concentrations of natalizumab were performed in every rabbit of each group (Group A: 0.625 mg, Group B: 1.25 mg, and Group C: 2.5 mg). Baseline electroretinogram (ERG) and fundus photography were performed prior to injection. At days 1, 7, and 21 postinjection, ERG and fundus photography of each eye were performed. At last follow-up, Group C animals with highest drug concentration were sacrificed and the enucleated eyes were evaluated for retinal toxicity using transmission electron microscopy (TEM). RESULTS: No difference in ERG responses was observed in eyes injected with low and intermediate concentration of natalizumab between day 0 and day 21. Furthermore, rabbits injected intravitreally with highest dose showed reduction in amplitude of "a" wave (P = 0.0017) and a reduction in amplitude of "b" wave of ERG at day 21 (P = 0.0117). TEM revealed changes in the outer plexiform layer and inner nuclear layer, suggestive of toxicity primarily to the photoreceptor synaptic terminals and bipolar cells. CONCLUSION: Low-dose (0.625 mg) and intermediate-dose (1.25 mg) intravitreal injection of natalizumab appears safe for rabbit retina. However, functional and anatomical changes were observed in rabbit retina following a high-dose (2.5 mg) intravitreal injection of a monoclonal antibody blocking α4ß1 integrin.

Un-segregated waste disposal: an alarming threat of antimicrobials in surface and ground water sources in Delhi.

Exposure of active pharmaceutical compounds (APCs) to the environment during human use is of potential importance in the emergence of drug resistance, changing soil microbiota and their residual effect on living organisms. Thus, this study aimed to assess the extent of exposure of APCs in the hydrologic cycle in and around New Delhi. This study analyzed the presence of 28 drugs from different classes in the surface water (river Yamuna) and aquifers collected from 48 places in Delhi (within the radius of 40 km). The collected water samples were quantified for APCs content using LC-MS/MS. This study revealed that aquifers are extensively affected in most areas based on the accumulation of APCs in water resources to the levels > 0.01 µg/L. Interestingly, a geographical plot of total APCs studied indicated clustering in aquifers with such high levels closer to an unscientific landfill. This 30-year-old un-segregated landfill is found to drain leachate into surface water that had high APCs. This study further revealed that apart from therapeutic usage, the main source of ecological exposure could be due to the disposal of unused and expired pharmaceutical compounds into landfills. For the first time, this study revealed the existence of antimicrobial agents and other APCs in the aquifers of Delhi with levels > 0.1 µg/L, which is a matter of serious concern in terms of multi-drug resistance and other environmental perils. This study warrants the enforcement of regulations for the disposal of unused/expired APCs in high-density population areas.

Circulating biomarkers in glaucoma, age-related macular degeneration, and diabetic retinopathy.

Biomarkers to predict the altering physiological conditions over the period leading toward the ocular disorders are of major importance in therapeutics. Isolation and validation of the biomarkers specific to ocular diseases are a challenging task. Glaucoma is a neurodegenerative disease of the eye where the correlation of biomarkers in circulating fluid may be made specific for the eye. However, conditions such as wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (DR), circulating biomarkers might be having some degree of overlap with other conditions like cancer where a common factor such as angiogenesis is involved. Diabetes, a systemic disorder affecting the target organs such as eye, kidney, heart, and nervous system can be predicted using common circulating biomarkers. However, these markers need to be validated along with various stages of disease progression to enable the possibility of targeted pharmacological interventions apart from good glycemic control alone. This review compiles the attempts made to correlate such circulating biomarkers in the ocular conditions such as glaucoma, AMD, and DR in the search for a surrogate marker for diagnostic and prognostic value. To make biomarkers for the common convenience, genetic markers are excluded from this review.

A novel immunodeficient NOD.SCID-rd1 mouse model of retinitis pigmentosa to investigate potential therapeutics and pathogenesis of retinal degeneration.

Retinitis pigmentosa (RP) is a common retinal degeneration disease caused by mutation in any gene of the photo transduction cascade and results in photoreceptor dystrophy. Over decades, several animal models have been used to address the need for the elucidation of effective therapeutics and factors regulating retinal degeneration to prohibit or renew the damaged retina. However, controversies over the immune privilege of retina during cell transplantation and the role of immune modulation during RP still remain largely uninvestigated because of the lack of suitable animal models. Here, we have developed an immunocompromised mouse model, NOD.SCID-rd1, for retinitis pigmentosa (RP) by crossing CBA/J and NOD SCID mice and selecting homozygous double mutant animals for further breeding. Characterization of the newly developed RP model indicates a similar retinal degeneration pattern as CBA/J, with a decreased apoptosis rate and rhodopsin loss. It also exhibits loss of T cells, B cells and NK cells. The NOD.SCID-rd1 model is extremely useful for allogenic and xenogenic cell-based therapeutics, as indicated by the higher cell integration capacity post transplantation. We dissect the underlying role of the immune system in the progression of RP and the effect of immune deficiency on immune privilege of the eye using comparative qPCR studies of this model and the immune-competent RP model.

Involvement of Renin-Angiotensin System in Retinopathy of Prematurity - A Possible Target for Therapeutic Intervention.

OBJECTIVE: Examining the Retinal Renin Angiotensin System (RRAS) in the ROP neonates and analyzing the possibility of modulating the RRAS to prevent the progression in Oxygen Induced Retinopathy (OIR) model. METHOD: Vitreous of ROP patients (n = 44, median age 5.5 months) was quantified for RRAS components, VEGF, HIF-1α and compared with age matched control. The involvement of RRAS in ROP was tested in the rat model of OIR and compared with normoxia. Expressions of RAS components, VEGF and HIF-1α in retina were analyzed using qPCR and retinal structure and function was also analyzed. Effect of Angiotensin Converting Enzyme Inhibitor (ACEI) and Angiotensin Receptor Blocker (ARB) was evaluated and compared with Bevacizumab which served as a positive control. Drug penetration into retina was confirmed by liquid chromatography coupled ESI-tandem mass spectroscopy (LC-MS/MS). RESULTS: Multifold increase in the expression of RAS components in human vitreous and rat retina showed their involvement in ROP. ERG & fundus studies in OIR revealed the altered function of retina and were successfully prevented by ARB (telmisartan), ACEI (lisinopril) and bevacizumab. Retinal analysis revealed the presence of ACEI and ARB in their therapeutic levels. CONCLUSION: This study for the first time demonstrates the upregulated level of RAS components in human ROP vitreous and further that the pharmacological intervention in RRAS can functionally and structurally preserve retina against the progression of ROP in the OIR model.

Evaluation of hepatic metabolism and pharmacokinetics of ibuprofen in rats under chronic hypobaric hypoxia for targeted therapy at high altitude.

With studies indicative of altered drug metabolism and pharmacokinetics (DMPK) under high altitude (HA)-induced hypobaric hypoxia, consideration of better therapeutic approaches has continuously been aimed in research for HA related illness management. DMPK of drugs like ibuprofen may get affected under hypoxia which establishes the requirement of different therapeutic dose regimen to ensure safe and effective therapy at HA. This study examined the effects of the chronic hypobaric hypoxia (CHH) on hepatic DMPK of ibuprofen in rats. Experimental animals were exposed to simulated altitude of 7620 m (∼25,000 ft) for CHH exposure (7 or 14 days) in decompression chamber and administered with ibuprofen (80 mg/kg, body weight, p.o.). Results demonstrated that CHH significantly altered PK variables of ibuprofen and activities of both phase-I and II hepatic metabolic enzymes as compared to the animals under normoxic conditions. Hepatic histopathological observations also revealed marked alterations. Increase in pro-inflammatory cytokines/chemokines viz. IL-1ß, IL-2, IFN-γ, TNF-α exhibited close relevance with diminished CYP2C9 expression under CHH. Moreover, the down-regulated CYP2C9 level further supported the underlying mechanism for reduced metabolism of ibuprofen and as a result, increased retention of parent drug in the system. Increased mean retention time, Vd, T½ of ibuprofen, and decreased AUC, Cmax and clearance during CHH further strengthened the present findings. In conclusion, CHH exposure significantly affects hepatic DMPK of ibuprofen, which may further influence the usual therapeutic dose-regimen. Further, there is requirement of human studies to evaluate their susceptibility toward hypobaric hypoxia.

Pharmacological Interventions for Vascular Targeting in Retinopathy of Prematurity: An Experimental Study.

Purpose: This study was conducted to evaluate the pharmacological interventions to target vascular proliferation in the Retinopathy of Prematurity (ROP). Methods: Protein Kinase C modulator (Bryostatin), tubulin polymerization inhibitor (Dolastatin 10), antiVEGF (Bevacizumab) and a non-specific VEGF inhibitor (Thalidomide) were screened in Retinopathy of Prematurity (ROP) model. The retinal vasculature was evaluated by calculating the tortuosity indices of vessels and electroretinography responses in terms of 'b' wave amplitude and was recorded from ROP rats on postnatal Day 17 and Day 25. Results: Retinopathy was seen in the form of tortousity of vessels at the posterior pole with arteries being affected more than veins. Maximum reduction in tortousity of vessels and the highest 'b' wave amplitude noted in bryostatin with a significant correlation between the two. Conclusion: Bryostatin showed a potential anti-angiogenic effect on the progression of ROP and may hold a promising future in the treatment of ROP.

Finger printing of counterfeit bevacizumab and identifying it before clinical use.

BACKGROUND: Bevacizumab is widely used for ophthalmic purposes. Recently, counterfeit bevacizumab has become a matter of concern. We analysed samples of suspected counterfeit formulations of bevacizumab and assessed the possibility of using simple tests in the clinic by ophthalmologists to prevent the use of counterfeit preparations in patients. METHODS: We did a protein analysis using Bradford assay and SDS-PAGE to confirm the presence of bevacizumab in 16 samples - 6 suspected and 10 others. The samples were also subjected to physicochemical analysis such as osmolarity, chloride content and pH. The samples tested negative for protein were analysed by mass spectrometry to detect drugs used in place of bevacizumab. We standardized the method of frothing and precipitation analysis for identifying authentic samples of bevacizumab before their clinical use. RESULTS: Five of the 16 samples tested were negative for the presence of bevacizumab. The physicochemical parameters also supported the protein analysis test. However, no ionizable organic compound (other drug[s]) was detected by mass spectrometry. CONCLUSION: Ophthalmic use of counterfeit bevacizumab can be prevented by simple methods such as the frothing and precipitation tests. These can identify the absence of an active drug.