Avoiding hindsight in non-obviousness determination: case law review of pharmaceutical patents and guidance from the KSR v Teleflex decision.

Introduction: Hindsight bias is the tendency to estimate an outcome once it is known. Legal systems are often prone to hindsight bias. In patent law, the non-obviousness or inventive step is the most critical determinant of patentability and often subjected to hindsight bias.Areas covered: Scholarly literature confirms the existence of hindsight bias in different patent systems. This communication hence addresses factors, which lead to hindsight bias specifically in chemical and pharmaceutical arts, guidance from the case law that can be helpful in avoiding hindsight bias in non-obviousness determination.Expert opinion: The Supreme Court in 2007, advocated a more expansive and flexible approach to where the Teaching Suggestion or Motivation test could come from. In the case of chemical and pharmaceutical active compounds, the considerations such as i) was there sufficient motivation to modify the lead compound and arrive at the claimed compound and its properties, ii) was there a reasonable expectation of success to achieve the claimed property and other such considerations highlighted in this review may contribute to avoid hindsight bias in non-obviousness determination.

Strigolactones and their crosstalk with other phytohormones.

BACKGROUND: Strigolactones (SLs) are a diverse class of butenolide-bearing phytohormones derived from the catabolism of carotenoids. They are associated with an increasing number of emerging regulatory roles in plant growth and development, including seed germination, root and shoot architecture patterning, nutrient acquisition, symbiotic and parasitic interactions, as well as mediation of plant responses to abiotic and biotic cues. SCOPE: Here, we provide a concise overview of SL biosynthesis, signal transduction pathways and SL-mediated plant responses with a detailed discourse on the crosstalk(s) that exist between SLs/components of SL signalling and other phytohormones such as auxins, cytokinins, gibberellins, abscisic acid, ethylene, jasmonates and salicylic acid. CONCLUSION: SLs elicit their control on physiological and morphological processes via a direct or indirect influence on the activities of other hormones and/or integrants of signalling cascades of other growth regulators. These, among many others, include modulation of hormone content, transport and distribution within plant tissues, interference with or complete dependence on downstream signal components of other phytohormones, as well as acting synergistically or antagonistically with other hormones to elicit plant responses. Although much has been done to evince the effects of SL interactions with other hormones at the cell and whole plant levels, research attention must be channelled towards elucidating the precise molecular events that underlie these processes. More especially in the case of abscisic acid, cytokinins, gibberellin, jasmonates and salicylic acid for which very little has been reported about their hormonal crosstalk with SLs.

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families.

Plant-derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke-water (SW) and the smoke-derived compounds, karrikinolide (KAR1 ) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke-water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10(-6) and 10(-7) m) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10(-3) m) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant-derived smoke and the smoke-isolated compounds may stimulate pollen growth in a wide range of plant species.

Polyaspartic acid functionalized gold nanoparticles for tumor targeted doxorubicin delivery.

In this paper, we present polyaspartic acid, a biodegradable polymer as a reducing and functionalizing agent for the synthesis of doxorubicin loaded gold nanoparticles by a green process. Gold nanoparticles were stable to electrolytes and pH. Secondary amino groups of polyaspartic acid enabled reduction of gold chloride to form gold nanoparticles of size 55 +/-10 nm, with face centered cubic crystalline structure as confirmed by UV, TEM, SAED and XRD studies. Cationic doxorubicin was readily loaded onto anionic polyaspartic acid gold nanoparticles by ionic complexation. Fluorescence studies confirmed doxorubicin loading while FTIR spectra confirmed ionic complexation. Doxorubicin loading onto polyaspartic acid gold nanoparticles was studied at doxorubicin/polyaspartic acid molar ratios 1:10 to 1:1. As the molar ratio tended to unity, although loading up to 60% was achieved, colloidal instability resulted and is attributed to effective covering of negative charges of polyaspartic acid. Stable doxorubicin loaded polyaspartic acid gold nanoparticles of 105 +/- 15.1 nm with doxorubicin loading of 23.85% w/w and zeta potential value of -28 +/- 0.77 mV were obtained at doxorubicin/polyaspartic acid molar ratio 1:10. Higher doxorubicin release rate from the doxorubicin loaded polyaspartic acid gold nanoparticles in an acid medium (i.e., pH 5.5) as compared to that in pH 7.4 and deionized water is a desirable characteristic for tumor targeted delivery. Enhanced cytotoxicity and 3 fold higher uptake of doxorubicin loaded polyaspartic acid gold nanoparticles as compared to doxorubicin solution were seen in MCF-7 breast cancer cells while polyaspartic acid gold nanoparticles revealed no cytotoxicity confirming safety. Prominent regression in tumor size in-vivo in fibrosarcoma tumor induced mouse model was observed upto 59 days with doxorubicin loaded polyaspartic acid gold nanoparticles while doxorubicin solution treated mice showed regrowth beyond 23rd day. Moreover, a decrease of body weight of 35% indicating severe toxicity with doxorubicin solution as compared to only 20% with gradual recovery after day 30 in case of doxorubicin loaded polyaspartic acid gold nanoparticles confirmed their lower toxicity and enhanced efficacy.

Anti-inflammatory and mutagenic evaluation of medicinal plants used by Venda people against venereal and related diseases.

ETHNOPHARMACOLOGY RELEVANCE: Inflammation is a major risk factor for various human diseases including venereal diseases, often resulting in treatment complications. Plants have been traditionally used for treatment of many different diseases and have been successfully proven to be an alternative source in treatment of infectious diseases. AIM OF THE STUDY: This study was aimed at evaluating the anti-inflammatory activities and the mutagenic properties of 12 medicinal plants used by the Venda people against venereal and related diseases. MATERIALS AND METHODS: The plants were evaluated for their anti-inflammatory activity against the cyclooxygenase (COX-1 and -2) enzymes and genotoxicity using the Ames test, with and without S9 (metabolic activation) against Salmonella typhimurium tester strain TA98. RESULTS: DCM and PE extracts of Adansonia digitata bark, Bolusanthus speciosus bark, Pterocarpus angolensis bark and Pappea capensis leaves and EtOH and water extracts of Bolusanthus speciosus stem and Ekebergia capensis bark showed the best anti-inflammatory activity in both COX-1 and -2 assays at 250 µg/ml. These were further evaluated at three other concentrations (31.25, 62.5, and 125 µg/ml) to determine IC50 values. Water extracts of Ekebergia capensis bark showed the best IC50 value towards COX-1. The Ames test revealed that all plant extracts were non-mutagenic towards Salmonella typhimurium strain TA98 except for Elephantorrhiza burkei and Ekebergia capensis that showed weak mutagenicity. CONCLUSION: The active plants may offer a new source of chemicals for the effective treatment of anti-inflammatory conditions related to venereal diseases.

Hereditary ADAMTS 13 deficiency presenting as recurrent acute kidney injury.

We report here a case of 26-year-old male who presented with history of recurrent acute renal failure associated with microangiopathic hemolytic anemia and thrombocytopenia. ADAMTS 13 deficiency due to mutation in the gene encoding for ADAMTS 13 was identified as the cause. After eight episodes of acute kidney injury (AKI), patient started developing hypertension, proteinuria, and renal insufficiency. Treatment with regular monthly plasma infusions prevented further episodes of AKI and stabilized the renal function. Hypertension and proteinuria are controlled with angiotensin II receptor blockers.

Pharmacological properties and protein binding capacity of phenolic extracts of some Venda medicinal plants used against cough and fever.

ETHNOPHARMACOLOGY RELEVANCE: Several ailments are caused by infectious bacteria and in other diseases; they act as co-infection which complicate human life by causing health hazards. In Venda (South Africa), many plants are used in traditional medicine to treat cough and fever. AIM OF THE STUDY: This study was aimed at evaluating the antibacterial and antifungal properties, cyclooxygenases (COX), acetylcholinesterase (AChE) enzyme inhibitory effects and the phenolic composition as well as mutagenic properties of six medicinal plants used by the Venda people of Limpopo Province of South Africa against cough and fever. MATERIALS AND METHODS: The petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water extracts of six plants were tested against four infectious bacteria (Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus) and a fungus Candida albicans. The same extracts were evaluated for their ability to inhibit COX-1 and -2 enzymes. Methanolic and water extracts of the same plant were tested for acetylcholinesterase inhibitory effects. Total phenolics, flavonoids, gallotannins and condensed tannins were determined. The ability of the extracts to bind and precipitate proteins was also investigated. The extracts were investigated for genotoxicity with and without S9 (metabolic activation) against three Salmonella typhimurium tester strains TA98, TA100 and TA102. RESULTS: The organic extracts of Rhus lancea leaves exhibited the best antibacterial activity with minimum inhibitory concentration (MIC) values ranging from 0.0061 to 0.049mg/ml. The best antifungal activity was observed from a DCM extract of Syzygium cordatum leaves with a MIC value of 0.195mg/ml. The methanolic and water extracts of the same plant exhibited high inhibitory effects towards AChE with IC(50) values of 0.22 and 0.26mg/ml, respectively. The highest levels of flavonoids and gallotannins were detected in Spirostachys africana bark; 11.57 and 48.88µg/g, respectively. The highest percentages (1.2%) of condensed tannins were detected in Uvaria caffra leaves. The high levels of phenolic compounds may have been responsible for high antimicrobial activities for extracts of S. africana bark and U. caffra leaves. S. cordatum leaves represented the highest affinity for protein binding with 93%. All the extracts were non-mutagenic towards the three tested strains with and without S9 metabolic activation. CONCLUSION: The result obtained in this study goes a long way in validating the ethnobotanical usage of these medicinal plants in the treatment of cough and fever by the Venda people. However, more evidence obtainable from other assays not performed here are urgently required to confirm these results.

Antimicrobial properties and phenolic contents of medicinal plants used by the Venda people for conditions related to venereal diseases.

ETHNOPHARMACOLOGY RELEVANCE: Many people are infected by venereal diseases and the human immunodeficiency virus (HIV) in rural areas. Sexual transmitted diseases are considered a disgrace in the community because of the stigmas attached to them. Indigenous people tend to use several medicinal plants to treat these infectious diseases rather than western medicines. AIM OF THE STUDY: This study was aimed to evaluate the antibacterial, antifungal, antigonococcal, HIV-type 1 reverse transcriptase (RT) and to determine phenolic content of twelve medicinal plants used by the Venda people to treat venereal diseases. MATERIALS AND METHODS: The dried plant materials of twelve medicinal plants were extracted with petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water. The extracts were evaluated for their antimicrobial properties against two Gram-positive (Bacillus subtilis and Staphylococcus aureus), three Gram-negative (Neisseria gonorrhoeae, Escherichia coli and Klebsiella pneumoniae) bacteria and a fungus Candida albicans. The phenolic contents including total phenolics, flavonoids, gallotannins and condensed tannins of the methanolic extracts of the same plants were also determined. RESULTS: DCM and EtOH extracts of Bolusanthus speciosus bark and stems exhibited good activity (<0.1mg/ml) against Escherichia coli and Staphylococcus aureus with MIC values ranging from 0.098 to 0.012 mg/ml. The DCM extracts of Bolusanthus speciosus bark showed the best activity against Candida albicans with MIC and MFC values of 0.012 mg/ml. Water and 50% methanol extracts of Acacia karroo bark, Bolusanthus speciosus stems and Ximenia caffra roots and leaves showed good RT inhibition percentages (>70%) at 1mg/ml. All tested extracts exhibited dose dependent IC(50) values ranging from (0.1 to 0.6 mg/ml). Almost all plant species investigated contained phenolic compounds, which were greater than 5mg/g with the exception of Adansonia digitata bark and Aloe chabaudii roots. The highest level of flavonoids (11.9 µg/g) were detected in Ximenia caffra leaves. Whereas the highest amount of gallotannins were detected in Ekebergia capensis bark (69 µg/g). Condensed tannins were higher in Ekebergia capensis bark and Ximenia caffra roots (0.47 and 0.48% respectively). Adansonia digitata bark and Aloe chabaudii roots exhibited low levels of phenolic compounds as well as antimicrobial activities. CONCLUSION: The presented results offer supporting evidence for effective use of selected plant extracts. More in vitro confirmatory tests using other assays and/or in vivo tests are required. Toxicity studies are urgently needed to support the safe usage of these plants.

Management of severe extra-articular contracture of the elbow by open arthrolysis and a monolateral hinged external fixator.

Arthrolysis and dynamic splinting have been used in the treatment of elbow contractures, but there is no standardised protocol for treatment of severe contractures with an arc of flexion < 30 degrees . We present our results of radical arthrolysis with twin incisions with the use of a monolateral hinged fixator to treat very severe extra-articular contracture of the elbow. This retrospective study included 26 patients (15 males and 11 females) with a mean age of 30 years (12 to 60). The mean duration of stiffness was 9.1 months (5.4 to 18) with mean follow-up of 5.2 years (3.5 to 9.4). The mean pre-operative arc of movement was 15.6 degrees (0 degrees to 30 degrees ), with mean pre-operative flexion of 64.1 degrees (30 degrees to 120 degrees ) and mean pre-operative extension of 52.1 degrees (10 degrees to 90 degrees ). Post-operatively the mean arc improved to 102.4 degrees (60 degrees to 135 degrees ), the mean flexion improved to 119.1 degrees (90 degrees to 140 degrees ) and mean extension improved to 16.8 degrees (0 degrees to 30 degrees ) (p < 0.001). The Mayo elbow score improved from a mean of 45 (30 to 65) to 89 (75 to 100) points, and 13 had excellent, nine had good, three had fair and one had a poor result. We had one case of severe instability and one wound dehiscence which responded well to treatment. One case had deep infection with poor results which responded well to treatment. Our findings indicate that this method is very effective in the treatment of severe elbow contracture; however, a randomised controlled study is necessary for further evaluation.

Variation in heavy metals and microelements in South African medicinal plants obtained from street markets.

South African medicinal plants are traditionally harvested from a wide range of undisclosed locations by plant gatherers. Thus, there is a risk that plant material may be exposed to a variety of pollutants. The variation in five heavy metals (arsenic, cadmium, cobalt, nickel, and lead) and six essential elements (boron, copper, iron, manganese, molybdenum, and zinc) was determined in commonly used South African medicinal plants obtained from street markets. Elemental content was determined using inductively coupled plasma optical emission spectrophotometry. The reliability of the procedure was ensured by analysing a certified reference material. Medicinal plant samples contained arsenic and cadmium at levels exceeding the World Health Organization limits of 1 and 0.3 mg kg(-1) respectively. Lead and nickel were detected in all samples. Elevated iron and manganese levels were recorded in certain plant species. Multiple metal contamination of parts of medicinal plants gives grounds for concern. This study emphasizes the unsafe consequences of the South African practice of collecting medicinal plants from undisclosed locations and making these readily available to the public.

Toxicity of metal elements on germination and seedling growth of widely used medicinal plants belonging to Hyacinthaceae.

In South Africa, pollution of agricultural soils is on the increase primarily due to excessive application of fertilizers, sewage disposal and mining activities. This study was done to determine the effect of trace elements (Cu, Zn) and heavy metals (Cd, Pb, Hg) on germination and seedling development of Bowiea volubilis, Eucomis autumnalis and Merwilla natalensis. These medicinal plant species are highly recommended for cultivation in South Africa to reduce the pressure on wild populations. Copper and Zn at 1 mg L(-1) significantly (p < 0.05) reduced the percentage germination of E. autumnalis. Low concentrations (>or=1 mg L(-1)) of Cu and Zn negatively affected the roots of all three species. Mercury concentrations of 0.5 and 1 mg L(-1) significantly (p < 0.05) decreased the percentage germination of B. volubilis and E. autumnalis respectively. Cadmium and Hg at 2 mg L(-1) showed a detrimental effect on the root growth of B. volubilis. Concentrations of 0.5 mg L(-1) of all heavy metals tested significantly (p < 0.05) decreased shoot length of M. natalensis.

Mechanistic investigations of phase behavior in Eudragit E blends.

Blends of Eudragit E (EE) and polymeric excipients using thermal analysis and FTIR spectroscopy were examined. The interactions amongst the blend components were quantified in terms of parameters K1 and K2 in Schneider equation and were explained on the basis of interactions between the functional groups of the blend constituents investigated by FTIR spectroscopy. EE formed miscible blends with EC and polyelectrolyte complexes increasing in strength in the order: ES

Improving seedling vigour of indigenous medicinal plants with smoke.

The application of smoke and aqueous smoke solutions stimulates seed germination in a number of plant species. This study highlights the effects of aerosol smoke and smoke solutions on the germination and seedling vigour of three South African indigenous medicinal plants Albuca pachychlamys, Merwilla natalensis and Tulbaghia violacea. The vigour index of one-week-old seedlings of all three species examined was increased with the application of dry smoke and smoke extract dilutions, as compared to control treatments. Seedlings of A. pachychlamys germinated with smoke solutions showed a significant (p0.05) gain in bulb and leaf mass (27.9 and 197.6 mg respectively) compared to untreated seedlings (9.9 and 124.7 mg respectively) when grown in vitro for 75 days. The leaf mass of smoke solution-treated seedlings of T. violacea was significantly (p0.05) higher (120.4 mg) than that of untreated seedlings (47.6 mg). Subsequently, the height of seedlings in both species was also significantly (p0.05) greater. Seedlings germinated in water and then transferred to smoke solutions (1:2000) showed enhancement of some of the growth parameters studied. Albuca pachychlamys and T. violacea seeds exposed to aerosol smoke exhibited higher seedling survival percentages than from non-smoked seeds, while no significant effect was observed for M. natalensis seedlings. This investigation shows that the application of smoke technology can be adopted to produce high vigour seedlings.

Enhanced capacities and selectivities for cholesterol in aqueous media by molecular imprinting: role of novel cross-linkers.

Molecularly imprinted polymers are being increasingly investigated as selective sorbents. For the recovery of cholesterol from aqueous media, the utility of the molecularly imprinted polymers has been limited by modest capacities and selectivities, especially when compared with alternative adsorbents reported for the binding of bile acids [Macromolecules 34 (2001) 1548]. This paper describes the use of cholesterol conjugated monomers and cross-linkers, which bind to the template cholesterol molecule by hydrophobic interactions. This leads to enhanced capacities and selectivities during the recovery of cholesterol from aqueous media. The templating effect is clearly seen in the enhanced capacity and selectivity in the retention of cholesterol vis-a-vis stigmasterol and testosterone.

Morphological changes in degrading PLGA and P(DL)LA microspheres: implications for the design of controlled release systems.

The in vitro degradation of microspheres of polymers of lactic and glycolic acids were investigated by monitoring the mass loss from the device, the molecular weight of the polymer and the morphological changes of the particles with time. Two different sequences of morphological changes were found to be operative, depending upon the polymer from which they were made--one, (I) for the high molecular weight P(DL)LA, and the other, (II) for all PLGAs and the low molecular weight P(DL)LA. Microspheres of category I showed clear evidence of heterogeneous degradation, where the initially dense microsphere developed a hollow interior. Microspheres of category II plasticized on hydration due to reduction in the Tg of the polymer below the incubation temperature of 37 degrees C. There was suppression of release of entrapped globular proteins from microspheres that underwent plasticization (category II), while slow and sustained release was seen from those that did not (category I). It is proposed that plasticization renders the matrix of category II microspheres non-porous, which prevents release by pore-diffusion. The mass loss profiles of PLGA were found to be different from those reported in the literature, in that the rates of mass loss after an initial lag time were not as rapid as has been reported. The experimental conditions used, namely the use, or otherwise, of agitation, is suggested as the reason for these differences and the need to draw a correlation between in vitro experimental conditions and in visa behaviour is emphasized.

Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers.

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.

Seroprevalence of human immunodeficiency virus infection in an infectious disease hospital.

AIMS AND OBJECTIVES: To analyse clinicopathological features of HIV infected patients admitted in an infectious disease hospital in Mumbai. MATERIAL AND METHODS: Retrospective study of 501 patients admitted from 1st September, 1996 to 28th February, 1998 and screened for HIV status out of clinical suspicion was carried out. HIV seropositivity was established by double confirmation of spot test results with microwell Elisa test. RESULTS: HIV seropositivity was seen in 39.92%. Out of these 94% were adults with male preponderance and 96.5% had only HIV-1 infection. The significant clinical features in HIV positive patients were chronic diarrhea (51.5%), prolonged fever (41.5%) and history of exposure (34%). Pulmonary tuberculosis could be diagnosed in 19.5% of HIV positive patients. In patients with prolonged/recurrent jaundice HBsAg and HIV was noted as a coinfection within 10/42 cases tested. CONCLUSIONS: Increasing prevalence of HIV seropositivity was noted in patients admitted to an infectious disease hospital. Association of HIV infection with tuberculosis and in icteric cases with HBsAg was significant. The HIV screening should be carried out in patients with prolonged illness resistant to usual mode of treatment.

Molecularly imprinted adsorbents for positional isomer separation.

2,4-Dihydroxybenzophenone (2,4-DHB) imprinted polymers were synthesized by surface imprinting technique, using allyl alcohol as the functional monomer. The polymers showed a very high selectivity for 2,4-DHB when compared with various positional isomers such as 2-HB, 2,2'-DHB, 4,4'-DHIB and 4,4'-DMB. Solvents were found to affect the selectivity as well as sorption capacity in the case of surface imprinted polymers. The selectivities decreased drastically when the imprint cavity was blocked. This validated the importance of the cavity and the rebinding interactions in governing the selectivity in the case of MIPs. The surface imprinted polymers also showed a high selectivity under non-equilibrium conditions thereby making them suitable adsorbents for industrial separations.

Preparation of non-porous microspheres with high entrapment efficiency of proteins by a (water-in-oil)-in-oil emulsion technique.

Emulsification-solvent removal methods have been widely used for encapsulating bioactive macromolecules like proteins and polypeptides in biodegradable polymers. We report, a (water-in-oil)-in-oil emulsion technique wherein proteins and polypeptides differing in molecular weight and shape were encapsulated in polymers of current biomedical interest. When an oil was used as the processing medium in combination with a carefully selected mixed solvent system such that a stable (w/o1/o2 emulsion is formed and solvents are removed by a combination of extraction and evaporation, the entrapment efficiency was high and the product nonporous. The entrapment efficiency of globular proteins exceeded 90% while that of fibrous proteins was around 70%. Fracture studies revealed that the polymer matrix was dense. The mechanism of entrapment involved solvent-induced precipitation of the protein as the microspheres were being formed. The principle of the method will find use in preparation of non-porous polymer microparticles with reduced burst effect.