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Mefenamic acid (MA), a poorly water-soluble drug, was used as a model substance to investigate granules and tablet characteristics to be optimized for the loading volume of MA (0-74.1% v/v) in the formulation including lactose monohydrate/maize starch (7/3) as excipients. The compactibility of granules increased with loading volume of MA. This was related to the brittle behavior of MA during compression and the increase of intragranular pore volume of granules. The minimum disintegration time (266+/-8.3 s) was found in the tablet that was composed of 55.1% v/v MA and 13.6% v/v maize starch. The determination of the critical concentration of disintegrant (% v/v) required for a minimum disintegration time is suggested to be useful for solid dosage form design.

This study was performed to elucidate the influences of process and formulation design using power consumption and temperature measurements during granulation. Power consumption was recorded "in process" using a previously introduced computer program for optimal end-point detection at an early stage. The temperature increase (DeltaT) during granulation was recorded using a temperature sensor. The temperature increase in the wet powder bed expresses the friction forces at interparticle contacts occurring during granulation. The maxima of temperature profile occurred at 130% saturation, whereas the maxima of power consumption were determined at 100% saturation. The ratio of temperature and power consumption (TPR factor) is introduced as a signature of formulation design. TPR factor was found to be dependent on particle size, particle surface, water absorption capacity and solubility of the excipient and model drug, respectively. However, TPR factor was found to be independent of process design, such as the filling level of the mixer. Understanding and controlling the granulation process is a key factor in robust dosage form design. The "in process" control fits ideally the prerequisites of a drug quality system for the 21st century and FDA's Process Analytical Technology (PAT) initiative. The results of previous and present works of our research group will be used in a following step to develop an artificial neural network for granulation "in process" control.

Using the molecular-dynamics technique, cluster emission for 5 keV Ar bombardment of a Cu (111) surface has been investigated using a many-body (tight binding) potential for the Cu-Cu interaction. The calculations allow us to analyse the basic processes underlying cluster emission. It is found that two distinct processes can be distinguished which lead to cluster emission under energetic ion bombardment. The first process causes the emission of small clusters, which are emitted by a collective motion during the development of the collision cascade within the first picosecond after impact. Thus, emission times of such clusters agree with the emission times of atoms in sputtering. Such a process can be envisioned if, for example, a few layers below the surface, an energetic recoil causes the development of a subcascade. Energy transferred by this event to the surface is strongly directional and can lead to the simultaneous emission of a group of neighbouring surface atoms, which in some cases will remain bounded and form a cluster after emission. Typically, clusters emitted by this mechanism consist of atoms, which are neighbouring in the target and are almost exclusively surface atoms, similar to all sputtered atoms. Emission of large clusters (cluster sizes of 10 or more atoms), as observed experimentally, is a puzzling phenomenon. From our calculations we conclude that the emission of such large clusters does not occur during the collisional phase of sputtering, but happens much later (5-10 ps after ion impact). Emission can occur for spike events, where all the energy of the impinging ion is deposited locally in a small volume near to the surface, and the sputtering yield is 3-5 times the average yield. Such events are rare, but we have found a few cases in our calculations where stable clusters consisting of more than 20 atoms were emitted. Melting of the spike volume occurs, and the high temperatures and pressures produced can cause emission of large fragments during the thermal phase. The composition of such large clusters is quite different from that of small clusters. They consist of atoms from different layers and the constituents are also generally not next-neighbour atoms. This change in origin of the cluster atoms reflects the mixing and diffusion processes occurring in the melted zone before emission. The calculations indicate that hydrodynamical phenomena might play a role in the emission of large fragments. Additional calculations, where the energy was distributed 'thermally' in a three-dimensional volume under the surface for 500 fs, give very similar results, even in such cases where the kinetic phase of the collision-cascade development was absent.

A quasicontinuous granulation and drying process to avoid scale-up problems is introduced in this work. Consistent and reproducible granule quality is a key factor in robust dosage form design and fits ideally the prerequisites of a drug quality system for the twenty-first century and the Food and Drug Administration's Process Analytical Technology (PAT) initiative. In scale-up, factors that simulate or reproduce the laboratory scale must be considered. This system provides a new possibility for industrial manufacturing and galenical development of pharmaceutical solids. The quasicontinuous method described in the present work, and the laboratory and production batches and the granulating equipment used to produce them, are the same. Once a robust process has been defined in the laboratory, it is merely repeated as many times as necessary to achieve the desired final batch size. The quasicontinuous process gives new possibilities to simplify manufacturing procedures and to validate them faster. The quality of the resulting granules and tablets compared with classical methods is equal until better. In many cases, existing products have been transferred to the multicell process without formulation changes. The quasicontinuous production concept for high-shear granulation and fluid-bed drying offers many advantages over the classical methods used to produce pharmaceutical granules. The wet massing process may be monitored by the power consumption of the mixer motor for each subunit, as in classical high-shear granulation processes. The air volume, temperature, and humidity of each of the drying cells may be controlled individually to avoid overheating of temperature-sensitive materials. All processing variables must be precisely controlled by a computer, and the data must be collected for documentation. As such, product quality and reproducibility for each subunit is assured.

Objective: To determine if the number of diagnostic laparoscopies done on women without tubal adhesive disease could be reduced by testing for tubal disease with Chlamydia trachomatis antibody titers and hysterosalpingography (HSG), either singly or together., Design: Historical prospective chart review., Setting: The Colorado Kaiser Permanente Reproductive Endocrinology Clinic., Patients: All 703 infertility patients who had C. trachomatis antibody titers done from March 2, 1988 to April 30, 1992. The final study group was comprised of 218 patients who had antibody titers, HSG, and laparoscopy., Interventions: None., Main Outcome Measures: Sensitivity, negative predictive value, and false-positive rate were the test characteristics of interest. Tubal disease was identified by laparoscopy., Results: For HSG testing, the sensitivity was 78% and the negative predictive value was 85%. For C. trachomatis titers, the sensitivity was also 78% and the negative predictive value was 82%. Ninety-five percent confidence intervals for sensitivity and negative predictive value overlapped, indicating that there was no significant difference. However, false-negative rates were the same for the two tests, but false-positive rates were lowest for HSG and series testing., Conclusions: To minimize false-positive tests and thus, to minimize unnecessary laparoscopies, HSG testing either alone or combined with the C. trachomatis antibody titer as series tests yielded a significantly lower false-positive rate. In our study group, if both tests were negative, tubal disease was identified on laparoscopy in only 5% of cases. Choice of most cost-effective test sequence will depend on who bears the cost. Further studies of cost-benefit using well-defined testing sequences are needed to determine if C. trachomatis antibody titers in series with HSG would be more cost effective than HSG alone in detecting tubal disease.

Objective: To delineate the relationship between the pulsatile gonadotropin inputs in early follicular phase of the menstrual cycle and the P secretions by the corpus luteum in women., Design: For measuring pulsatile release of gonadotropin, blood samples were drawn every 15 minutes for 24 hours in the early follicular phase. Daily blood samples were drawn for LH, FSH, E2, and P., Setting: The reproductive endocrine unit of a university hospital., Patients: Fourteen patients with luteal phase defect (LPD) and 12 normally cycling women., Results: The length of follicular phase in LPD was significantly shorter than that of women with normal cycles. There were significant differences in LH pulsatile secretions and amplitudes in LPD patients when compared with those of women with normal cycles. Basal E2, PRL, and preovulatory E2 concentrations were not different between the two groups whereas the peak of P secretions in luteal phase was significantly decreased in LPD., Conclusions: These data suggest that LPD may result from the altered LH pulse frequency in early follicular phase of the menstrual cycle. Whether this increased LH pulse frequency results from an intrinsic disease of the pulse oscillator or to some event in the preceding cycle remains unknown. It is tempting to speculate that an increased LH pulsatile secretion in the early follicular phase of menstrual cycles in patients with LPD may down-regulate the LH secretion at midcycle, thereby lowering the LH surge, which in turn reduces the P secretion in luteal phase.

Objective: The role of histocompatibility antigens in pregnancy outcome is controversial. This controversy may be because the initial studies were of small numbers or the patient groups were not homogeneous. The purpose of this study is to clarify these discrepant results by carrying out histocompatibility antigen typing and mixed lymphocyte culture on couples with idiopathic recurrent spontaneous abortion and by comparing results with those of fertile couples., Study Design: Sixty couples with at least three spontaneous abortions and 60 normal couples with at least two successful pregnancies were included. Histocompatibility antigen typing and mixed lymphocyte culture were performed by using the standard techniques. The data were analyzed statistically with the Fisher exact test and chi 2., Results: Our results failed to show any difference between normal and aborting couples with regard to HLA-A, HLA-B, and HLA-DR distribution or sharing or to mixed lymphocyte culture responsiveness., Conclusion: Our study, along with other studies, emphasizes that the histocompatibility antigen system does not have an impact on pregnancy outcome.

Y-1 adrenal tumor cells were grown on plastic, or plastic treated with poly(2-hydroxyethyl methacrylate) (polyHEMA) to produce concentration-dependent rounding (10(-5)-3 X 10(-4) M) of the cells or on plastic treated with poly-D-lysine (polylysine) to produce flat cells, in order to determine whether or not cell shape is correlated with steroid synthesis. The degree of rounding of cells was measured by determining mean cell height and longest cell diameter. Three measurements of steroid production were made: production of 20 alpha-dihydroprogesterone, transport of cholesterol to the inner mitochondrial membrane, and production of pregnenolone by isolated mitochondria. Cells grown on poly(HEMA) showed increase in mean cell height, decrease in longest diameter (i.e. rounding), and increase in all three measurements of steroidogenesis. In the case of synthesis of 20 alpha-dihydroprogesterone, the response was dependent on the concentration of poly(HEMA), being greater with higher concentrations (up to 10(-4) M), of this agent. Moreover the degree of rounding (cell height) was correlated with production of 20 alpha-dihydroprogesterone at three concentrations of poly(HEMA) (r = 0.93. ACTH at a supramaximal concentration produced increases in all of these responses to the poly(HEMA) surface. Polylysine produced flatter cells (lower mean height and greater longest diameter) than plastic and also inhibited all three steroidogenic responses to ACTH. (Bu)2cAMP exerted the same effects as ACTH. Growing cells on poly(HEMA) or polylysine did not affect production of cyclic AMP by the cells. Addition of poly(HEMA) or polylysine to the medium in which the cells were incubated, at the same concentrations as those used for influencing cell shape, was without effect on steroid synthesis or the response to ACTH. Cells grown on poly(HEMA) show decreased incorporation of [3H] thymidine into DNA. It is concluded that cell shape influences the delivery of cholesterol to inner mitochondrial membrane and in this way, increases the production of steroids by Y-1 cells and that the effects of poly(HEMA) on cell shape, cholesterol transport, and synthesis of DNA may involve microfilaments.

An 18-year-old patient with Turner's syndrome presented with cyclical vaginal bleeding and spontaneous development of secondary sexual characteristics. She demonstrated classic features of Turner's phenotype, and a culture of blood lymphocytes revealed a 45,XO karyotype. The patient's plasma and urinary estrogen concentrations were similar to those in normal adult women in the late proliferative phase. In contrast, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were elevated to values seen in postmenopausal women. Dynamic testing revealed no estrogen response to human menopausal gonadotropin (/MG) and a paradoxical fall in estrogen after administration of human chorionic gonadotropin (hCG). Administration of luteinizing hormone-releasing hormone (LH-RH) resulted in an exaggerated response of both gonadotropins. Dilatation and curettage revealed endometrial hyperplasia, and a laparotomy revealed the presence of 2 gonadlike structures. The histologic diagnosis was lutein cyst. Karyotyping of lymphocytes, skin from the abdominal incision, and tissue from both gonadal structures revealed a 45,XO karyotype without evidence of mosaicism.

Three female siblings with isolated gonadotropin deficiency were studied. None demonstrated hyposmia or craniofacial abnormalities. Single doses of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone (TRH) produced markedly different responses within the kindred. Following administration of TRH, one subject displayed a sharp rise in prolactin, but thyrotropin did not change.

Evaluation of a method for predicting and confirming ovulation by measurement of vaginal and salivary electrical resistance (VER and SER) was the purpose of this study. Eighteen menstrual cycles from 13 subjects were analyzed. A clearly defined nadir in VER at day 0, the day of the luteinizing hormone (LH) peak, followed by a pronounced increase the following day was observed. A peak in SER was consistently observed 5 to 6 days before day 0. The correlation coefficient (r) between the cycle day of the SER peak and cycle day of the LH peak was 0.94. After the peak in SER, values were low for several days but increased 1 to 2 days before the LH peak. Results indicate that monitoring of SER and VER may provide the basis for a simple method for predicting and confirming ovulation.

The testicular enzyme, 17,20-lyase, catalyzes the removal of the C-17 side chain from steroids in the synthesis of androgens. This activity employs cytochrome P-450 as an oxygen donor. Attempts to purify the cytochrome and its reductase from testis microsomes have previously been unsuccessful due to the low concentrations of these components (2--5% that of liver). The cytochrome and reductase were solubilized from rat testis microsomes using a mixture of sodium cholate and Emulgen 913. The components were then separated by DEAE chromatography. The cytochrome was further purified by chromatography using hydroxylapatite for an 8.5-fold enrichment. The reductase was further purified by hydroxylapatite and affinity chromatography. An 84-fold enrichment was achieved. 17,20-Lyase activity could be partially restored by mixing the cytochrome and reductase in the presence of phospholipid.

A 24-year-old phenotypic girl with primary amenorrhea was referred for further evaluation. The cervix and uterus were absent and no gonadal tissue was present by physical examination or at laparotomy. Her serum FSH was elevated and there was no increase in serum testosterone following HCG stimulation. The defect(s) in this case and similar patients with "agonadism" reported to date are compatible with various genetic hypotheses.

A 25-year-old woman was seen for the complaint of secondary amenorrhea. Skull roentgenograms revealed a markedly enlarged sella turcica. Studies of pituitary and hypothalamic function including prolactin were normal. A pneumoencephalogram revealed dilated ventricles and a mass in the septum pellucidum and hypothalamus. Partial removal of this hypothalamic astrocytoma and placement of an interventricular shunt resulted in the return of menses. Recurrent obstruction a few months later resulted in headache, disorientation, and amenorrhea. A shunt was placed again, resulting in clearing of symptoms and a reestablishment of normal menstrual cyclicity. It appears that the occurrence of increased intracranial pressure rather than the hypothalamic tumor caused the amenorrhea.

NADPH-cytochrome P-450 reductase (EC 1.6.2.4), the enzyme involved in progesterone 17-hydroxylation, was purified to apparent homogeneity by detergent solubilization of the microsomal fractions of liver and testis from untreated rats. The enzymes from these two tissues were then compared with regard to several parameters. The liver and testicular reductases have the same subunit mol wt (79,000), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and have similar specific activity for cytochrome c reduction (48.1 and 58.2 mumol min-1 mg-1, respectively). The absolute flavin absorption spectra of liver and testicular reductase were very similar. The spectra of native, reduced, and oxidized reductases were characteristic of flavoprotein with two flavin groups. The Km values for NADPH during cytochrome reduction were shown to be the same, within experimental error (5.29 +/- 0.46 microM-1 for liver and 4.37 +/- 0.53 microM-1 for testis). Monospecific antiserum was prepared against both rat liver and testicular reductases. There were no antigenic differences demonstrated by immunological tests using either antibody preparation. Antiliver reductase antiserum was shown to inhibit testicular microsomal progesterone 17-hydroxylase activity by 70%. Testicular cytochrome c reductase activity was inhibited by 94%, and liver cytochrome c reductase activity was inhibited by 85%. Peptide maps of both forms of reductase isolated from immunoprecipitates showed very similar patterns after enzymatic proteolysis. These results indicate that microsomal NADPH-cytochrome P-450 reductase involved in steroid hydroxylations in untreated rat liver and testis could not be distinguished from each other by these methods and, therefore, are probably the same protein.

A case of male pseudohermaphroditism associated with 17 alpha-hydroxylase deficiency is reported in which the diagnosis was firmly established by gas chromatography-mass spectrometry. The patient was a 20-year-old genotypic male, phenotypic female who presented with primary amenorrhea, absence of body hair, and no breast development. She was hypertensive. Corticosterone and progesterone levels were very high and sex steroids were virtually absent. Gonadotropins were in the menopausal range. The results of radioimmunoassay were not diagnostic. Analysis of urine using gas chromatography-mass spectrometry revealed a striking absence of steroids with 17-oxygen function-nor were there any 18 or 19 carbon steroids. These results clearly establish a total deficiency of 17 alpha-hydroxylase activity.

The steroid metabolic profile on a patient with a suspected block in steroid biosynthesis was analyzed by gas chromatography and gas chromatography-mass spectrometry. The results of this work led to an interpretation of a block at the 17 alpha-hydroxylase step. Although the steroid metabolic profile was complex, we could not detect any steroids with a hydroxy moiety at position C-17. The use of gas chromatography-mass spectrometry, in this case, was the method of choice for a final diagnosis because of the confusion that resulted from other, more classical, forms of analysis. Data from our patient's sample are reported and compared to normals and to other cases where a block in 17 alpha-hydroxylase have been reported.

This study was undertaken to evaluate the usefulness of measuring salivary and vaginal electrical resistance in monitoring ovulation induced by clomiphene citrate (CC). Data from 28 cycles of 12 women treated with CC were compared with those of 18 cycles of 13 women who were ovulating spontaneously. Patterns of salivary readings in CC and spontaneous cycles were similar and showed a preovulatory peak 6.2 (mean) days before the luteinizing hormone peak. The trend in vaginal readings for CC cycles differed from that of spontaneous ovulations in that the values were depressed during and shortly after CC therapy. Occurrence of the periovulatory nadir and subsequent rise was common to both groups. The rise in vaginal readings CC cycles occurred within 24 hours of the luteinizing hormone peak in 88% of cases. Retrospective analysis showed that, based on this method, artificial insemination would have been timed appropriately in 25 of 27 cycles or in every cycle, depending on the protocol used. The results indicate that the method is equally useful for predicting and confirming ovulation in cycles in which CC was used to induce ovulation as it is in spontaneous cycles.

A number of innovations have recently been made in the area of tubal surgery. This report summarizes our experience with these techniques. Microtechniques improved our results markedly with tubal anastomosis. Pregnancy rates were over 50% in reversal of fimbriectomy, but much lower with undilated tubes closed by inflammatory processes. Use of low molecular weight dextran was without benefit in improving pregnancy rates. Salpingolysis without steroids or hydrotubation resulted in a pregnancy rate of 69%.

The effect of metyrapone on the activity of the steroid 17alpha-hydroxylase from rat testis was evaluated. A competitive pattern of inhibition was observed after analysis of data using a least mean squares computer analysis. The substrate for the hydroxylase induced a Type I difference spectrum in an active suspension of Triton treated microsomes. The magnitude of this spectral change was dependent on steroid concentration and was diminished by metyrapone. The effect of metyrapone was abolished at infinite steroid concentration. These results confirm the participation of cytochrome P-450 as a reactant in the 17alpha-hydroxylase reaction.

The cytochrome P-450 of gonadal microsomes is an integral component of the steroid converting enzymes, 17 alpha-hydroxylase and 17,20-lyase. Interaction of the steroid substrates with this cytochrome results in a shift in the Soret band as measured by difference spectroscopy. In these studies it is shown that in contrast to placental microsomal cytochrome P-450 which binds C19 steroids, testis microsomal cytochrome P-450 primarily binds C21 steroids. However, addition of a 17 alpha- methyl, 17 beta-acetate or a 17 beta-benzoate group to testosterone permits interaction. The addition of hydroxyl or methyl groups to other positions does not affect binding. The presence of multiple oxygen functions on C21 steroids, as in cortisol and corticosterone, precludes interaction. At least one oxygen function seems necessary for binding as 5 alpha- and 5 beta-pregnane do not bind whereas 20-deoxypregnenolone (5-pregnen-3 beta-ol) does bind. These findings indicate that factors in addition to hydrophobic interactions dictate the binding of steroid substrates to testis microsomal cytochrome P-450.