Development and evaluation of nanoemulsion and microsuspension formulations of curcuminoids for lung delivery with a novel approach to understanding the aerosol performance of nanoparticles.

Extensive research has demonstrated the potential effectiveness of curcumin against various diseases, including asthma and cancers. However, few studies have used liquid-based vehicles in the preparation of curcumin formulations. Therefore, the current study proposed the use of nanoemulsion and microsuspension formulations to prepare nebulised curcuminoid for lung delivery. Furthermore, this work expressed a new approach to understanding the aerosol performance of nanoparticles compared to microsuspension formulations. The genotoxicity of the formulations was also assessed. Curcuminoid nanoemulsion formulations were prepared in three concentrations (100, 250 and 500 µg/ml) using limonene and oleic acid as oil phases, while microsuspension solutions were prepared by suspending curcuminoid particles in isotonic solution (saline solution) of 0.02% Tween 80. The average fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD) of the nebulised microsuspension formulations ranged from 26% and 7.1 µm to 40% and 5.7 µm, for 1000 µg/ml and 100 µg/ml respectively. In a comparison of the low and high drug concentrations of the nebulised nanoemulsion, the average FPF and MMAD of the nebulised nanoemulsion formulations prepared with limonene oil ranged from 50% and 4.6 µm to 45% and 5.6 µm, respectively; whereas the FPF and MMAD of the nebulised nanoemulsion prepared with oleic acid oil ranged from 46% and 4.9 µm to 44% and 5.6 µm, respectively. The aerosol performance of the microsuspension formulations were concentration dependent, while the nanoemulsion formulations did not appear to be dependent on the curcuminoids concentration. The performance and genotoxicity results of the formulations suggest the suitability of these preparations for further inhalation studies in animals.

In-process rheometry as a PAT tool for hot melt extrusion.

Real time measurement of melt rheology has been investigated as a Process Analytical Technology (PAT) to monitor hot melt extrusion of an Active Pharmaceutical Ingredient (API) in a polymer matrix. A developmental API was melt mixed with a commercial copolymer using a heated twin screw extruder at different API loadings and set temperatures. The extruder was equipped with an instrumented rheological slit die which incorporated three pressure transducers flush mounted to the die surface. Pressure drop measurements within the die at a range of extrusion throughputs were used to calculate rheological parameters, such as shear viscosity and exit pressure, related to shear and elastic melt flow properties, respectively. Results showed that the melt exhibited shear thinning behavior whereby viscosity decreased with increasing flow rate. Increase in drug loading and set extrusion temperature resulted in a reduction in melt viscosity. Shear viscosity and exit pressure measurements were found to be sensitive to API loading. These findings suggest that this technique could be used as a simple tool to measure material attributes in-line, to build better overall process understanding for hot melt extrusion.

Application of hot melt extrusion for improving bioavailability of artemisinin a thermolabile drug.

Hot melt extrusion has been used to produce a solid dispersion of the thermolabile drug artemisinin. Formulation and process conditions were optimized prior to evaluation of dissolution and biopharmaceutical performance. Soluplus®, a low Tg amphiphilic polymer especially designed for solid dispersions enabled melt extrusion at 110 °C although some drug-polymer incompatibility was observed. Addition of 5% citric acid as a pH modifier was found to suppress the degradation. The area under plasma concentration time curve (AUC0-24h) and peak plasma concentration (Cmax) were four times higher for the modified solid dispersion compared to that of pure artemisinin.

A novel transflectance near infrared spectroscopy technique for monitoring hot melt extrusion.

A transflectance near infra red (NIR) spectroscopy approach has been used to simultaneously measure drug and plasticiser content of polymer melts with varying opacity during hot melt extrusion. A high temperature reflectance NIR probe was mounted in the extruder die directly opposed to a highly reflective surface. Carbamazepine (CBZ) was used as a model drug, with polyvinyl pyrollidone-vinyl acetate co-polymer (PVP-VA) as a matrix and polyethylene glycol (PEG) as a plasticiser. The opacity of the molten extrudate varied from transparent at low CBZ loading to opaque at high CBZ loading. Particulate amorphous API and voids formed around these particles were found to cause the opacity. The extrusion process was monitored in real time using transflectance NIR; calibration and validation runs were performed using a wide range of drug and plasticiser loadings. Once calibrated, the technique was used to simultaneously track drug and plasticiser content during applied step changes in feedstock material. Rheological and thermal characterisations were used to help understand the morphology of extruded material. The study has shown that it is possible to use a single NIR spectroscopy technique to monitor opaque and transparent melts during HME, and to simultaneously monitor two distinct components within a formulation.

Monitoring ibuprofen-nicotinamide cocrystal formation during solvent free continuous cocrystallization (SFCC) using near infrared spectroscopy as a PAT tool.

The purpose of this work was to explore NIR spectroscopy as a PAT tool to monitor the formation of ibuprofen and nicotinamide cocrystals during extrusion based solvent free continuous cocrystallization (SFCC). Drug and co-former were gravimetrically fed into a heated co-rotating twin screw extruder to form cocrystals. Real-time process monitoring was performed using a high temperature NIR probe in the extruder die to assess cocrystal content and subsequently compared to off-line powder X-ray diffraction measurements. The effect of processing variables, such as temperature and mixing intensity, on the extent of cocrystal formation was investigated. NIR spectroscopy was sensitive to cocrystal formation with the appearance of new peaks and peak shifts, particularly in the 4800-5200 cm(-1) wave-number region. PXRD confirmed an increased conversion of the mixture into cocrystal with increase in barrel temperature and screw mixing intensity. A decrease in screw rotation speed also provided improved cocrystal yield due to the material experiencing longer residence times within the process. A partial least squares analysis in this region of NIR spectrum correlated well with PXRD data, providing a best fit with cocrystal conversion when a limited range of process conditions were considered, for example a single set temperature. The study suggests that NIR spectroscopy could be used to monitor cocrystal purity on an industrial scale using this continuous, solvent-free process.

Simultaneous determination of cinnamaldehyde, eugenol and piperine by HPTLC densitometric method.

An HPTLC densitometric method for the simultaneous determination of cinnamaldehyde and eugenol as well as trace amounts of piperine in pepper-contaminated cinnamon was developed. The applicability of the method was tested with cinnamon bark powder adulterated with pepper powder, cinnamon oil, clove powder, clove oil and a commercial preparation containing cinnamaldehyde and eugenol. The method was validated for specificity, precision, accuracy and robustness. The method was found to be precise for different concentrations of cinnamaldehyde, eugenol and piperine. The accuracy of the method was checked by conducting a recovery study at three different levels. The linearity was found to be in the ranges 52.54-735.56, 533.2-8531.2 and 50-300 ng/spot, respectively, with correlation coefficients of 0.9985 +/- 0.04, 0.9982 +/- 0.06 and 0.9937 +/- 0.11 for cinnamaldehyde, eugenol and piperine.

Effect of rifampicin pretreatment on the transport across rat intestine and oral pharmacokinetics of ornidazole in healthy human volunteers.

Increased exsorption of ornidazole was observed from different parts of the small intestine of the rat after pretreated with rifampicin and sodium butyrate by the everted sac method. Based on the in vitro studies the effect of rifampicin pretreatment on the pharmacokinetics of ornidazole was investigated in eight healthy male volunteers. After an overnight fast, 500 mg ornidazole was administered to the volunteers, either alone or after 6 days pretreatment with a once daily dose of 600 mg rifampicin. Serum concentrations of ornidazole were estimated by reverse phase HPLC. Pharmacokinetic parameters were determined based on non-compartmental model analysis using the computer program Win Nonlin 1.1. Rifampicin preteatment resulted in a significant decrease in AUC, C(max) and t1/2, by 21.16%, 20.43% and 18.11%, respectively. Clearance was increased significantly by 32.14%. This may be due to increased induction of cytochrome P450 enzymes and/or increased expression of P-glycoprotein. This interaction may have clinical significance when ornidazole is co-administered with rifampicin in chronic treatment conditions, such as tuberculosis, leprosy and other infections of joints, bones, etc.

Effect of system variables involved in packed column supercritical fluid chromatography of stavudine taken as model analyte using response surface methodology along with study of thermodynamic parameters.

A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of stavudine analysis by packed column supercritical fluid chromatography (PC-SFC). The effect of simultaneously varying the pressure, temperature and modifier concentration was studied to optimize the method in order to obtain excellent chromatographic figures of merit. The method is based on isocratic elution using methanol-modified supercritical carbon dioxide as the mobile phase at the flow rate of 3.0 ml/min through a JASCO Finepak SIL-5, ODS [C(18) (5 microm, 25 cm x 4.6 mm, i.d.)] column support using photodiode array detection. The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. From the response surface graphs optimum regions were selected to be +1, -1, and +1 for temperature (60 degrees C), pressure (20 MPa) and percent modifier concentration (17.81%, v/v), respectively. Linearity dynamic range was found to be in the range of 2.0-150.0 microg/ml with significantly high value of correlation coefficient. The method was validated for precision, robustness and recovery to assess the viability of the established method. The chromatographic limit of detection and quantitation were 0.80 and 1.50 microg/ml respectively. The method has been successfully used to analyze commercial dosage form to assess the chromatographic performance of SFC system which was found to be 99.91%+/-1.62. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of stavudine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.

Effect of system variables involved in packed column SFC of nevirapine as model analyte using response surface methodology: application to retention thermodynamics, solute transfer kinetic study and binary diffusion coefficient determination.

A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of nevirapine analysis by packed column supercritical fluid chromatography (PC-SFC). The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. The method is based on methanol-modified carbon dioxide as the mobile phase at flow rate of 3.0 ml/min with elution through a JASCO Finepak SIL-5, [C18 (5-micron, 25 cm x 4.6 mm, i.d.)] column using photodiode array detection. The method has been successfully used to analyze commercial solid dosage form to assess the chromatographic performance of SFC system. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of nevirapine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.

The ICH guidance in practice: stress degradation studies on indinavir sulphate and development of a validated specific stability-indicating HPTLC assay method.

A sensitive, selective, precise and stability-indicating high-performance thin layer chromatography (HPTLC) method for analysis of indinavir sulphate both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon tetrachloride/chloroform/methanol/10% v/v ammonia (4:4.5:1.5:0.05, v/v/v/v). Densitometric analysis of indinavir sulphate was carried out in the absorbance mode at 260 nm. This system was found to give compact spots for indinavir sulphate (Rf value of 0.43 +/- 0.02, for six replicates). Indinavir sulphate was subjected to acid and alkali hydrolysis, oxidation, dry and wet heat treatment, and photo degradation. The drug undergoes degradation under acidic and basic conditions, oxidation, dry and wet heat treatment, and photo degradation. Also the degraded products were well resolved from the pure drug with significantly different Rf values. The method was validated for linearity, precision, robustness, limit of detection (LOD), limit of quantitation (LOQ), specificity and accuracy. Linearity was found to be in the range of 100-6000 ng/spot with significantly high value of correlation coefficient r2 = 0.997 +/- 0.64. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.999 +/- 0.002 in the working concentration range of 1000-6000 ng/spot. The LOD and LOQ were 40 and 120 ng/spot, respectively. Statistical analysis proves that the method is repeatable and specific for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of acid degradation process. Arrhenius plot was constructed and activation energy was calculated.

HPTLC method for guggulsterone. I. Quantitative determination of E- and Z-guggulsterone in herbal extract and pharmaceutical dosage form.

A sensitive, selective, precise and robust high-performance thin-layer chromatographic method of analysis of E and Z stereoisomers of guggulsterone (the hypolipidemic agent in the gum-resin exudates of Commiphora mukul) both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-acetone (9:1, v/v). Densitometric analysis of guggulsterone was carried out in the absorbance mode at 250 nm. This system was found to give compact spots for E- and Z-guggulsterone (Rf value of 0.38 +/- 0.02 and 0.46 +/- 0.02, respectively) following double development of chromatoplates with the same mobile phase. The linear regression analysis data for the calibration plots for E- and Z-guggulsterone showed good linear relationship with r2 = 0.9977 +/- 0.054 and 0.9975 +/- 0.068, respectively, in the concentration range of 100-6000 ng/spot. The mean value of slope and intercept were 0.11 +/- 0.006 and 0.11 +/- 0.005, 14.26 +/- 0.56 and 10.92 +/- 0.76, respectively, for E- and Z-guggulsterone. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 12, 10 and 24, 20 ng/spot, respectively, for E- and Z-guggulsterone. Statistical analysis proves that the method is repeatable and selective for the estimation of the said drug. Since the proposed mobile phase effectively resolves the E- and Z-isomers of guggulsterone, this HPTLC method can be applied for identification and quantitation of these isomers in herbal extracts and pharmaceutical dosage form.

HPTLC method for guggulsterone. II. Stress degradation studies on guggulsterone.

Stress degradation studies were carried out on guggulsterone (the hypolipidemic agent in the gum-resin exudates of Commiphora mukul) following the conditions prescribed in the parent drug stability testing guideline (Q1AR) issued by International Conference on Harmonization (ICH). The present study describes degradation of guggulsterone under different ICH prescribed stress conditions (acid and base hydrolysis, oxidation, dry and wet heat degradation and photodegradation) and establishment of a stability indicating HPTLC assay. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-acetone (9:1, v/v). Densitometric analysis of guggulsterone was carried out in the absorbance mode at 250 nm. This system was found to give compact spots for E- and Z-guggulsterone, (Rf value of 0.38 +/- 0.02 and 0.46 +/- 0.02, respectively) following double development of chromatoplates with the same mobile phase. The drug undergoes degradation under acidic and basic conditions, oxidation, dry and wet heat treatment and photodegradation. All the peaks of degraded products were resolved from the standard guggulsterone with significantly different Rf values. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.

Five additional genes are involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for beta-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

HPTLC method for determination of nevirapine in pharmaceutical dosage form.

A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis of nevirapine both as a bulk drug and in formulations was developed and validated. The solvent system consisted of toluene-carbon tetrachloride-methanol-acetone-ammonia (3.5:3.5:2.0:1.0:0.05, v/v/v/v/v). Densitometric analysis of nevirapine was carried out in the absorbance mode at 289nm. This system was found to give compact spots for nevirapine (R(f) value of 0.44+/-0.02). Nevirapine was subjected to acid and alkali hydrolysis, oxidation, dry heat and wet heat treatment and photodegradation. The drug undergoes degradation under acidic, basic conditions and oxidation. Also the degraded products were well resolved from the pure drug with significantly different R(f) values. Linearity was found to be in the range of 30-1000ng/spot with significantly high value of correlation coefficient. The linear regression analysis data for the calibration plots showed good linear relationship with r(2)=0.998+/-0.002 in the working concentration range of 300ng/spot to 1000ng/spot. The mean value of slope and intercept were 0.073+/-0.005 and 36.78+/-1.50, respectively. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 5 and 10ng/spot, respectively. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of acid degradation process. Arrhenius plot was constructed and activation energy was calculated.

Stability indicating HPTLC determination of linezolid as bulk drug and in pharmaceutical dosage form.

A simple, selective, precise, and stability-indicating high-performance thin layer chromatographic method of analysis of Linezolid both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-acetone (5:5, v/v). This system was found to give compact spots for Linezolid (Rf value of 0.29 +/- 0.01). Linezolid was subjected to acidic, alkali hydrolysis, oxidation, and photodegradation. The degraded products also were well separated from the pure drug. Densitometric analysis of Linezolid was conducted in the absorbance mode at 254 nm. The linear regression data for the calibration plots showed good linear relationship with r2 = 0.997 +/- 0.001 in the concentration range of 300-800 ng/spot. The mean value of correlation coefficient, slope, and intercept were 0.998 +/- 0.003, 0.15 +/- 0.009, and 19.52 +/- 1.66 respectively. The method was validated for precision, accuracy, ruggedness, and recovery. The limits of detection and quantification were 20 ng/spot and 50 ng/spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and photo degradation. All the peaks of degraded product were resolved from the standard drug with significantly different Rf values. This indicates that the drug is susceptible to acid-base hydrolysis, oxidation, and photo degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. Because the method could effectively separate the drug from its degradation products, it can be used as a stability indicating one.

Stability-indicating HPTLC determination of tizanidine hydrochloride in bulk drug and pharmaceutical formulations.

A simple, selective, precise and stability-indicating high-performance thin-layer chromatographic method of analysis of tizanidine hydrochloride both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-acetone-ammonia (5:5:0.1, v/v/v). This system was found to give compact spots for tizanidine hydrochloride (R(f) value of 0.32+/-0.01). Tizanidine hydrochloride was subjected to acid and alkali hydrolysis, oxidation and photodegradation. Also, the degraded product was well separated from the pure drug. Densitometric analysis of tizanidine hydrochloride was carried out in the absorbance mode at 315 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r(2)=0.9922 in the concentration range 300-1000 ng per spot. The mean value of correlation coefficient, slope and intercept were 0.9922+/-0.002, 0.064+/-0.001 and 38.09+/-1.71, respectively. The method was validated for precision, recovery and robustness. The limits of detection and quantitation were 88 and 265 ng per spot, respectively. The drug does not undergo degradation under acidic and basic conditions. The samples degraded with hydrogen peroxide showed additional peak at R(f) value of 0.12. This indicates that the drug is susceptible to oxidation. Statistical analysis proves that the method is repeatable and selective for the estimation of said drug. As the method could effectively separate the drug from its degradation product, it can be employed as a stability-indicating one.

Preparation and characterization of glassy celecoxib.

Celecoxib, a poorly water-soluble drug, was converted into a glassy state by melt quenching. The properties of glassy celecoxib were studied using infrared (IR) spectroscopy, differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), intrinsic dissolution rate (IDR), and thin-layer-chromatography (TLC). Glass transition occurred at 51.8 degrees C. Infrared spectrum of glass has revealed significant changes due to H-bonding. Celecoxib glass shows around 15 times faster dissolution as compared with the crystalline state. Heckel plot analysis has shown better compressibility in glassy state. Unpulverized glass remained stable for 3 months, whereas after pulverization about 70% crystallinity was gained in 100 hours. Further attempts may be carried out to stabilize the glass.

Stability indicating HPTLC determination of clopidogrel bisulphate as bulk drug and in pharmaceutical dosage form.

A sensitive, selective, precise and stability indicating high-performance thin layer chromatographic method of analysis of clopidogrel bisulphate both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of carbon tetrachloride-chloroform-acetone (6:4:0.15, v/v/v). This system was found to give compact spots for clopidogrel bisulphate (R(f) value of 0.30+/-0.01). Clopidogrel bisulphate was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also the degraded products were well separated from the pure drug. Densitometric analysis of clopidogrel bisulphate was carried out in the absorbance mode at 230 nm. The linear regression data for the calibration plots showed good linear relationship with r(2)=0.999+/-0.001 in the concentration range of 200-1000 ng. The mean value of correlation coefficient, slope and intercept were 0.999+/-0.001, 0.093+/-0.011 and 8.83+/-0.99, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 40 and 120 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of degraded product were resolved from the standard drug with significantly different R(f) values. This indicates that the drug is susceptible to acid-base hydrolysis, oxidation and dry heat degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.

Spherical crystallization of celecoxib.

Celecoxib exhibits poor flow properties and compressibility. Spherical crystallization of celecoxib was carried out using the solvent change method. An acetone:dichloromethane (DCM):water system was used where DCM acted as a bridging liquid and acetone and water as good and bad solvent, respectively. Hydroxypropylmethylcellulose (HPMC) was used to impart strength and sphericity to the agglomerates. The effect of amount of bridging liquid and speed of agitation was studied using 3(2) factorial design. Primary properties of the agglomerates were evaluated by infrared spectroscopy, powder X-ray diffraction, and differential scanning calorimetry. The effect of variables on micromeritic, mechanical, compressional, and dissolution behavior was evaluated by response surface methodology. Particle size, bulk density, mean yield pressure (MYP), and drug release were found to be significantly affected by either of the two variables. Interaction of variables significantly affected the MYP.

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production.

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.